Magnetic capture of a target from a fluid

ABSTRACT

Disclosed herein is an improved method for magnetic capture of target molecules (e.g., microbes) in a fluid. Kits and solid substrates for carrying the method described herein are also provided. In some embodiments, the methods, kits, and solid substrates described herein are optimized for separation and/or detection of microbes and microbe-associated molecular pattern (MAMP) (including, e.g., but not limited to, a cell component of microbes, lipopolysaccharides (LPS), and/or endotoxin).

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a divisional application of co-pending U.S.application Ser. No. 16/414,168 filed May 16, 2019, which is acontinuation application of U.S. application Ser. No. 15/522,686 filedApr. 27, 2017, which is a 371 National Phase Entry of InternationalPatent Application No. PCT/US2015/057516 filed on Oct. 27, 2015 whichclaims benefit under 35 U.S.C. § 119(e) of the U.S. ProvisionalApplication No. 62/068,912 filed Oct. 27, 2014, the contents of all ofwhich are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

The invention was made with Government Support under Contract Nos.N66001-11-1-4180 and BR0011-13-C-0025, both awarded by the United StatesDepartment of Defense/DARPA. The government has certain rights in theinvention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 26, 2017, isnamed 002806-082582-US SL.txt and is 18,045 bytes in size.

TECHNICAL DISCLOSURE

Embodiments of various aspects described herein relate to methods,compositions, and kits for magnetic capture of a target molecule (e.g.,cells, microbes, small molecules, chemicals, drugs, proteins, and/ornucleic acids) from a fluid, including bodily fluids such as blood,food, water, and environmental sources.

BACKGROUND

One of the disadvantages of conventional magnetic capture methods isthat the magnetic separation is based on relatively weak magnetic fieldgradients, which in turn provide limited effectiveness, for example, inseparating smaller magnetic particles from a fluid (e.g., a solution).For example, the existing magnetic capture methods and systems aregenerally limited to magnetic beads greater than 1 micrometer indiameter. In addition, the relatively weak magnetic field gradientlimits the size of the tube and the volume of fluid that can beprocessed. For example, DynaMag2™ (Invitrogen, Grand Island, N.Y.) isdesigned to work with magnetic beads greater than 1 micrometer indiameter and is thus not effective with smaller magnetic beads, such asthose in the 50 and 500 nanometer diameter range.

Many methods have been used to generate increased magnetic flux densitygradients (Kang et al., Small 3, 1784-1791 (2007); and Xia et al.,Biomed Microdevices 8, 299-308 (2006)), for example, using variousmicroelectromechanical system (MEMS) technologies, but they requirelabor-intensive and time-consuming fabrication processes for structuringferromagnetic materials at the nanometer to micrometer scale, such asphotolithography, LIGA(Lithographie-Galvanoformung-Abformung/Lithography-Electroplating-Molding),and CMP (chemical mechanical polishing). The MACS magnetic column(Miltenyi®) (Miltenyi et al., Cytometry 11, 231-238 (1990)) can be usedto trap smaller (e.g., 50 nm) magnetic particles. However, the MACSsystems use steel wool and/or magnetizable wires packed into a column toaccomplish magnetic gradient enhancement. However, the use of steel wooland/or magnetizable wires in a column makes the system harder to washcaptured cells, prone to clogging, and/or prone to inducing clottingwhen used with blood. In addition, the throughput of the MACS systems isvery limited (0.5 mL per a column). Due to the confined structures ofthe steel wool, it is difficult to apply the system to variousexperimental conditions or sample containers, such as tube or well plateconfigurations, fluidic devices (including microfluidic devices), etc.Accordingly, there is a need to develop novel and versatile methods,kits, devices, and systems for efficient and/or high throughput magneticseparation and/or capture of at least one or more target molecules froma fluid or solution.

SUMMARY

While smaller magnetic particles are more efficient in binding a widerrange of target molecules, efficient removal of small magnetic particlesfrom a fluid is challenging due to their low magnetic moments. Aspectsdescribed herein stem from, at least in part, discovery that forming a2D or 3D micro- or nano-structure of magnetic field concentratingparticles on a magnetic capture surface or a fluid-contact surface of amagnetic separation chamber, prior to introducing a fluid sample(comprising magnetic particles, e.g., target-binding magnetic particles)into the chamber to undergo magnetic separation, significantly enhancesmagnetic separation efficiency of the magnetic particles (e.g.,target-binding magnetic particles), even when the magnetic moments ofthe magnetic particles are too low to be removed by the existingmagnetic separation methods. During magnetic separation, the magneticfield concentrating particles are magnetized by an externally appliedmagnetic field, and substantially aligned with magnetic flux lines ofthe magnetic field to form a 2D or 3D micro- or nano-structure on amagnetic capture surface or a fluid-contact surface of a magneticseparation surface, thereby increasing or concentrating the magneticfield or flux density gradient locally experienced by magnetic particlesin a fluid, as compared to the magnetic field or flux density gradientwithout the magnetic field concentrating particles. Such magneticseparation method can be used to separate or capture at least one ormore (e.g., at least two or more) targets from a fluid when magneticparticles are adapted or functionalized to specifically bind thetarget(s).

In particular, inventors have demonstrated inter alia that the magneticseparation efficiency of microbes (e.g., S. aureus) bound to smallmagnetic beads (e.g., 50 nm or 128 nm in diameter) increasedsignificantly from 15%-30% to at least 95% or higher, when thefluid-contact surface of a microfluidic device channel was dispersedwith ferromagnetic particles forming a 2D or 3D micro- ornano-ferromagnetic structure thereon, prior to introducing a fluid to becleansed for magnetic separation. Additionally, the inventors have usedsuch method to effectively remove pathogenic contaminants from cordblood.

The concept of forming a 2D or 3D micro- or nano-structure of magneticfield concentrating particles on a fluid-contact surface can be extendedto magnetic separation of any target using appropriate target-bindingmagnetic particles in a wide range of separation device formats, e.g.,for static or continuous flow. Thus, the inventors have developed anovel, versatile and cost-effective method for increasing the magneticflux density gradient in a magnetic particle-based separation device ofany format (e.g., tube, multi-well plate, and/or microfluidic channels),and hence improving magnetic separation efficiency of a target moleculefrom a fluid. Accordingly, aspects described herein relate to methods,kits, devices, and compositions for sensitive magnetic separation orcapture of at least one or a plurality of (e.g., at least two or more)target molecules from a fluid. The methods, kits, devices, andcompositions described herein can be used for various applicationsincluding cleansing biological fluids as well as food, water, culturemedium (e.g., for pharmaceutical manufacturing or brewing), or any otherliquid.

One aspect described herein relates to a method of separating magneticparticles from a fluid. The method comprises: (a) subjecting a magneticcapture surface and magnetic field gradient concentrating particles to amagnetic field gradient (a gradient of a magnetic field), wherein themagnetic field gradient concentrating particles, in the presence of themagnetic field gradient, distribute on at least a portion of a magneticcapture surface and substantially align along magnetic flux lines of themagnetic field; and (b) contacting the magnetic capture surface with afluid comprising magnetic particles, wherein the magnetic field gradientconcentrating particles act as local magnetic field gradientconcentrators. At least a portion of the magnetic particles areattracted to the magnetic field gradient concentrating particles in thepresence of the magnetic field gradient, thereby separating the magneticparticles from the fluid. Due to enhancement of the magnetic fieldgradient by magnetic field gradient concentrating particlessubstantially aligning along with magnetic flux lines of a magneticfield applied to the method, such method is particularly useful forseparation of small magnetic particles with a magnetic moment that istoo low to be removed by the existing magnetic separation methods.

When the magnetic particles are functionalized to specifically bind atarget, the target-binding magnetic particles can be added to a fluidfor capture or separation of the target, if present, from the fluid.Accordingly, another aspect described herein relates to a method ofcapturing, removing, or separating one or more (e.g., at least two ormore) targets from a fluid. The method comprises introducing a fluid andtarget-binding magnetic particles to a magnetic separation chamber inthe presence of a magnetic field gradient (a gradient of a magneticfield), wherein at least a portion of a fluid-contact surface of themagnetic separation chamber comprises magnetic field gradientconcentrating particles distributed thereon and substantially alignedalong magnetic flux lines of the magnetic field. The magnetic fieldgradient concentrating particles act as local magnetic field gradientconcentrators, thus attracting at least a portion (e.g., at least 70% ormore) of the target-binding magnetic particles to the magnetic fieldgradient concentrating particles in the presence of the magnetic fieldgradient. Target(s) bound on the target-binding magnetic particles canthen be captured, removed, or separated from the fluid.

In some embodiments of this aspect and other aspects described herein,the target-binding magnetic particles can be added to the fluid, priorto introducing the mixture to the magnetic separation chamber, in whicha magnetic field gradient can be applied. Thus, the target(s) areallowed to bind to the target-binding magnetic particles, prior toexposing the mixture to a magnetic field gradient.

In some embodiments of this aspect and other aspects described herein,the fluid or fluid sample and the target-binding magnetic particles canbe added to a sample chamber or an open-top magnetic separation chamberwithout any magnetic field gradient therein. A structure comprising afluid-contact magnetic capture surface and magnetic field gradientconcentrating particles distributed on thereon can then be introducedinto the sample chamber or the open-top magnetic separation chamber sothat the fluid-contact magnetic capture surface is contacted with themixture comprising the fluid and the target-binding magnetic particlescontained in the sample chamber. The magnetic field gradientconcentrating particles distributed on the fluid-contact magneticcapture surface are substantially aligned along magnetic flux lines of amagnetic field (e.g., generated within the structure or appliedexternally to the structure).

In some embodiments of this aspect and other aspects described herein,the magnetic field gradient concentrating particles form magnetic micro-or nano-structures on at least a portion of the fluid-contact surface ofthe magnetic separation chamber. The magnetic micro or nano-structurescan be two dimensional or three dimensional.

In some embodiments of this aspect and other aspects described herein,at least 50% area or higher of the fluid-contact surface comprises themagnetic field gradient concentrating particles distributed thereon.

In some embodiments of this aspect and other aspects described herein,the magnetic field gradient concentrating particles can comprisesuperparamagnetic particles, paramagnetic particles, ferrimagneticparticles, ferromagnetic particles, or combinations thereof. In oneembodiment, the magnetic field gradient concentrating particles areferromagnetic particles. In one embodiment, ferromagnetic particles areparticles of reduced iron, atomized iron, electrolyte iron, orcombinations thereof.

In some embodiments of this aspect and other aspects described herein,the magnetic field gradient concentrating particles by themselves arenot able to bind or capture a target. In some embodiments of this aspectand other aspects described herein, the magnetic field gradientconcentrating particles do not comprise metal oxide (e.g., iron oxide).In some embodiments, the magnetic field gradient concentrating particlescan be treated to reduce non-specific interaction with a target to beremoved or separated from a fluid, e.g., by coating the surface of themagnetic field gradient concentrating particles with a blocking agent.Non-limiting examples of a blocking agent include a lubricant (e.g., butnot limited to silicone and/or mold-release agent), a polymer (e.g., butnot limited to silicon-based polymer such as polydimethylsiloxane(PDMS)), milk proteins, bovine serum albumin, blood serum, whole blood,and a combination of two or more thereof.

The magnetic field gradient concentrating particles can be larger,comparable to, or smaller than the target-binding magnetic particles insize. In some embodiments of this aspect and other aspects describedherein, the diameter of the magnetic field gradient concentratingparticles ranges from about 50 nm to about 5 mm. In one embodiment, thediameter of the magnetic field gradient concentrating particles is about300 μm.

The methods of various aspects described herein can be applied tomagnetic particles (e.g., target-binding magnetic particles) of variousmaterials and/or sizes, including magnetic particles with weak magneticmoments or small magnetic particles (e.g., nanoparticles). In someembodiments, the magnetic particles (e.g., target-binding magneticparticles) are particles of paramagnetic and/or superparamagneticmaterials. In one embodiment, the magnetic particles (e.g.,target-binding magnetic particles) are used in the methods describedherein. In some embodiments, the diameter of the magnetic particles(e.g., target-binding magnetic particles) is no more than 250 nm, nomore than 100 nm, no more than 50 nm, or no more than 5 nm.

The magnetic field gradient concentrating particles locally increasemagnetic flux density gradient when they are exposed to a magneticfield. Thus, the methods described herein increase the efficiency ofremoving magnetic particles from a fluid and thereby increasing theefficiency of magnetically capturing one or more target(s) from thefluid that is bound on targeting-binding magnetic particles. Theincrease in the efficiency can be at least about 50% (including, e.g.,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%) or more, as compared to the efficiency in the absence of themagnetic field concentrating particles. In some embodiments, theefficiency of magnetically capturing the target-bound targeting-bindingmagnetic particles from the fluid can be increased by at least about1.1-fold (including, e.g., at least about 1.5-fold, at least about2-fold, at least about 3-fold, at least about 4-fold) or more, ascompared to the efficiency in the absence of the magnetic fieldconcentrating particles.

The methods of various aspects described herein can be amenable to awide range of magnetic separation devices in various configurations.Thus, the magnetic separation chamber can comprise a channel, amicrofluidic channel, a sample well, a microtiter plate, a slide (e.g.,a glass slide), a flask (e.g., a tissue culture flask), a tube, ananotube, a fiber, a filter, a membrane, a scaffold, an extracorporealdevice, a mixer, a hollow fiber, or any combinations thereof. In someembodiments, the method described herein can be used in non-fluidicdevices (e.g., any sample carriers such as tubes with one open end, andmulti-well plates). Alternatively, the method described herein can beused in a fluidic device that allows a fluid flowing therethrough. Inthis embodiment, the fluid can flow through the magnetic separationchamber at a flow rate of about 1 ml/hr to about 10 L/hr.

Fluids of any sources can be introduced into the magnetic separationchamber. For example, the fluid can be a biological fluid obtained orderived from a subject, a fluid or specimen obtained from anenvironmental source, a fluid from a cell culture, a microbe colony, orany combinations thereof. In one embodiment, the fluid is a biologicalfluid selected from blood, plasma, cord blood, serum, lactationproducts, amniotic fluids, sputum, saliva, urine, semen, cerebrospinalfluid, bronchial aspirate, bronchial lavage aspirate fluid,perspiration, mucus, liquefied stool sample, synovial fluid, peritonealfluid, pleural fluid, pericardial fluid, lymphatic fluid, tears,tracheal aspirate, a homogenate of a tissue specimen, or any mixturesthereof. In one embodiment, the fluid is a fluid or specimen obtainedfrom an environmental source selected from a fluid or specimen obtainedor derived from food products, food produce, poultry, meat, fish,beverages, dairy product, water (including wastewater), ponds, rivers,reservoirs, swimming pools, soils, food processing and/or packagingplants, agricultural places, hydrocultures (including hydroponic foodfarms), pharmaceutical manufacturing plants, animal colony facilities,beer brewing, or any combinations thereof.

Methods and compositions for forming target-binding magnetic particlesare known in the art. In some embodiments, target-binding molecules canbe attached to magnetic particles via at least one or more linkersdescribed herein. In one embodiment, the linker is a peptidyl linker. Anexemplary peptidyl linker is an immunoglobulin or a portion thereof(e.g., but not limited to an Fc portion of an immunoglobulin).

The target-binding magnetic particles are magnetic particles adapted tospecifically bind a target molecule of interest. Example targetmolecules that can be captured or removed from a fluid include, withoutlimitation, cells, proteins, nucleic acids, microbes, small molecules,chemicals, toxins, drugs, and combinations thereof.

In one embodiment, the target-binding magnetic particles are adapted tospecifically bind a microbe (referred to as “microbe-binding magneticparticles”). The microbe-binding magnetic particles comprise on theirsurface microbe-binding molecules. Exemplary microbe-binding moleculefor use in the microbe-binding magnetic particles are opsonins, lectins,antibodies and antigen binding fragments thereof, proteins, peptides,peptidomimetics, carbohydrate-binding proteins, nucleic acids,carbohydrates, lipids, steroids, hormones, lipid-binding molecules,cofactors, nucleosides, nucleotides, nucleic acids, peptidoglycan,lipopolysaccharide-binding proteins, small molecules, and anycombination thereof.

In some embodiments, the microbe-binding molecule comprises at least amicrobial-binding portion of C-type lectins, collectins, ficolins,receptor-based lectins, lectins from the shrimp Marsupenaeus japonicas,non-C-type lectins, lipopolysaccharide (LPS)-binding proteins,endotoxin-binding proteins, peptidoglycan-binding proteins, or anycombinations thereof. In some embodiments, the microbe-binding moleculesis selected from the group consisting of mannose-binding lectin (MBL),surfactant protein A, surfactant protein D, collectin 11, L-ficolin,ficolin A, DC-SIGN, DC-SIGNR, SIGNR1, macrophage mannose receptor 1,dectin-1, dectin-2, lectin A, lectin B, lectin C, wheat germ agglutinin,CD14, MD2, lipopolysaccharide-binding protein (LBP), limulus anti-LPSfactor (LAL-F), mammalian peptidoglycan recognition protein-1 (PGRP-1),PGRP-2, PGRP-3, PGRP-4, C-reactive protein (CRP), or any combinationsthereof.

In some embodiments, the microbe-binding molecule is selected from thegroup consisting of MBL (mannose binding lectin), FcMBL (IgG Fc fused tomannose binding lectin), AKT-FcMBL (IgG Fc-fused to mannose bindinglectin with the N-terminal amino acid tripeptide of sequence AKT(alanine, lysine, threonine)), and any combination thereof.

In some embodiments, the microbe-binding molecule comprises an aminoacid sequence selected from the group consisting of: SEQ ID NO. 1, SEQID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ IDNO. 7, SEQ ID NO. 8, and any combinations thereof.

Another aspect described herein relates to a kit comprising (i) a devicecomprising a magnetic separation chamber or a magnetic capture surface;(ii) one or more containers containing magnetic field gradientconcentrating particles; and (iii) one or more containers containingtarget-binding magnetic particles.

In some embodiments, the device can further comprise a structure ormodule that can produce a magnetic field. In some embodiments thestructure or module that can produce a magnetic field can be detachablefrom the device, e.g., the magnetic separation chamber or magneticcapture surface.

The device comprising a magnetic separation chamber or a magneticcapture surface can be any fluid container or fluid processing device.For example, the device can be an eppendorf tube, a multi-well plate, aflask (e.g., a tissue culture flask), an extracorporeal device, a mixer,a hollow fiber cartridge, a microfluidic device, or any combinationsthereof. In some embodiments, the device is a microfluidic device. Inone embodiment, the device can be an organ-on-chip device (e.g., abiospleen device).

A solid substrate comprising a surface having magnetic field gradientconcentrating particles distributed thereon and substantially alignedalong magnetic flux lines of a magnetic field is also described herein.The solid substrate further comprises a target-binding magnetic particleand a target.

In some embodiments, the solid substrate can further comprise astructure or device that produces a magnetic field. Thus, the magneticfield gradient concentrating particles can be substantially alignedalong magnetic flux lines of the magnetic field produced by thestructure or device.

In one embodiment, the target is bound to the target-binding magneticparticle.

In some embodiments, the solid substrate is selected from the groupconsisting of a channel, a microfluidic channel, a sample well, amicrotiter plate, a slide (e.g., a glass slide), a flask (e.g., a tissueculture flask), a tube, a nanotube, a fiber, a filter, a membrane, ascaffold, an extracorporeal device, a mixer, a microfluidic device, ahollow fiber, or any combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G show diagrammatic views of ferromagnetic particle-integrateddevices according to various embodiments described herein. The devicewas used in the experiments described in the Examples herein to removemicrobes or microbe-associated molecular patterns (MAMP) from a fluid.FIG. 1A shows a diagrammatic scheme of an example ferromagneticparticle-integrated microfluidic device (e.g., biospleen device). Tocreate such a device, ferromagnetic particles suspended in buffer aretrapped in the channel by pumping the solution through the device withpermanent magnets attached. While one permanent magnet on one side ofthe device can be used, placing permanent magnets on both sides of thedevice significantly increased the magnetic flux density gradientsaround the ferromagnetic particles trapped in the channel. FIG. 1B showsa view of an example microfluidic device (e.g., biospleen device)without ferromagnetic particles. The biospleen device is a microfluidicdevice 100 that comprises two adjacent channels (source channel 140 andcollection channel 150) that are connected to each other by a series oftransfer channels 160: the source channel 140 contains flowing fluid tobe cleansed, e.g., blood, and the collection channel 150 has a bufferedsolution that collects and removes the target molecules that travelthrough the transfer channels 160. In one embodiment, the device 100comprises a central body 110 and outer layers 120 and 130. A fluid orfluid sample can flow into the source channel 140 through one or moreinlet ports 142 and exits the device 100 through one or more outletports 144. A collection fluid can flow into the collection channel 150through one or more inlet ports 152 and exits the device 100 through oneor more outlet ports 154. See the International Patent Application No.WO 2012/135834, the content of which is incorporated herein by referencein its entirety, for additional description of the biospleen device.Other microfluidic devices of different designs can also be employed.FIG. 1C shows a cross-sectional view of the device in FIG. 1B that isused in combination with ferromagnetic particles to enhance magneticseparation efficiency. In one embodiment, the device 100 comprises asource channel 140, a collection channel 150, and a plurality oftransfer channels 160 connecting the source channel 140 and thecollection channel 150. While the transfer channels 160 are shownoriented substantially perpendicular to the source channel 140 andcollection channel 150, the transfer channels 160 can be oriented in arange of angles (e.g., 1 to 90 degrees, where 0 degrees corresponds tothe direction of flow in the source channels 140) with respect to thesource channel 140. One or more magnetic sources 410, such as a magnet,can be positioned in close proximity to the collection channel 150. Whena magnetic source 410 is positioned closer to a fluid-contact surface151 of the collection channel 150, ferromagnetic particles 170 aredistributed on the fluid-contact surface 151 of the collection channel150. FIG. 1D shows a cross-sectional view of the device 100 in FIG. 1Bwith two magnetic sources 410 (e.g., magnets) placed facing each other,where a magnetic source 410 (e.g., a magnet) is placed in closerproximity to the collection channel 150 and another magnetic source 410(e.g., another magnet) is placed in closer proximity to the sourcechannel 140 (left panel), and a schematic diagram showing distributionof magnetic flux lines generated by the two magnets (right panel). InFIG. 1D, the ferromagnetic particles substantially align along themagnetic flux lines. FIG. 1E is a photograph of a ferrofluid in amagnetic field showing normal-field instability caused by a magnetplaced beneath a dish. A ferrofluid is a colloidal liquid comprisingnanoscale ferromagnetic, or ferrimagnetic, particles suspended in acarrier fluid (usually an organic solvent or water). FIG. 1F is aphotograph showing an example distribution of magnetic flux lines basedon one embodiment of magnet arrangement. FIG. 1G is a schematic diagramshowing various sizes of ferromagnetic particles (e.g., nano- ormicro-sized ferromagnetic particles) aggregating and aligning alongmagnetic flux lines. In some embodiments, smaller ferromagneticparticles can preferentially aggregate and align along magnetic fluxlines at a higher magnetic flux density. In some embodiments, largerferromagnetic particles can preferentially aggregate and align alongmagnetic flux lines at a lower magnetic flux density. Accordingly, insome embodiments of various aspects described herein, the magnetic fieldgradient concentrating particles utilized in the methods describedherein can comprise a mixture of different sized magnetic field gradientconcentrating particles. A mixture of different sized magnetic fieldgradient concentrating particles can be used when the magnetic fieldgradient is not uniform.

FIG. 2 is a line graph showing correlation of the magnetic isolationefficiency with different amounts of the ferromagnetic particles trappedin a microfluidic device (e.g., a biospleeen device). S. aureus (10⁴cfu/mL) bound with 128 nm FcMBL magnetic beads in TBST Ca⁺⁺ were flowedthrough a channel (e.g., of the biospleen device) primed with theferromagnetic particles (300 mg-900 mg) at a flow rate of 2 L/h.

FIG. 3 is a line graph showing isolation efficiency of S. aureusprebound with 50 nm FcMBL magnetic beads in TBST buffer (10 mL) in asingle round of magnetic separation using the methods described herein.The “biofluid” or “sample” channel (e.g., of the biospleen device) wasprimed with the ferromagnetic particles (500 mg) and the permanentmagnets applied the magnetic fields from the top and bottom of thedevice, which greatly enhanced the magnetic forces acting on the 50 nmmagnetic beads that have weak magnetic moment.

FIG. 4 is a table showing the results of blood culture vials inoculatedwith cord blood samples (250 uL) that has been treated in aferromagnetic particles-integrated microfluidic device (e.g., biospleendevice) for an indicated time period (T0=0 hour; T2=2 hours; T4=4 hours;and T5=5 hours). The microbe removal efficiency was determined based onthe turbidity of the inoculated blood culture vials after a 5-dayculture at 37° C. The “0” symbol indicates the 250 μL cord blood sampledid not contain pathogen while the “X” symbol indicates that the 250 μLcord blood sample contained at least one pathogen.

FIG. 5 is a bar graph showing RS218 E. coli depletion efficiency using50 nm FcMBL magnetic beads and ferromagnetic particles. Because ofhighly enhanced magnetic flux density gradients generated byferromagnetic beads, RS218 captured by 50 nm beads were efficientlyremoved whereas the conventional Dynal magnetic rack yielded less than5% depletion efficiency.

FIGS. 6A-6D show application of the methods described herein in variousmulti-well ELISA platform (e.g., 96-well plate-based ELISA platform).FIG. 6A shows the cross-sectional view of the KingFisher deep well plateworking with bar magnets and ferromagnetic particles. Integration offerromagnetic particles with the KingFisher system enhances magneticcapturing efficiency even when 50 nm magnetic beads were used to capturetarget species, e.g., microbes such as pathogens. FIG. 6B shows that theferromagnetic particle-integrated 96 well plates can capture andsignificantly deplete 50 nm magnetic beads when combined with a magneticplate holder and a shaker. FIG. 6C shows another embodiment of aferromagnetic particle-integrated 96-well plate system. A multiwellplate (e.g., 96 well plate) with ferromagnetic particles added in eachwell is brought in close proximity to or in contact with an array ofmagnets. During magnetic separation, the multiwell plate can be rotatedto facilitate the mixing. The system can be used to pull down 50 nmmagnetic beads (e.g., bound with target species such as pathogens)efficiently. FIG. 6D is a bar graph comparing the depletion efficiencyof RS218 E. coli bound on 50 nm microbe-binding magnetic particles(e.g., FcMBL-coated magnetic beads) in different 96-well plate-basedplatform (in shown in FIGS. 6A-6C) using ferromagnetic particles. Theconventional method without ferromagnetic particles was also performedas a control. All three different capture platforms yielded over 90%depletion efficiency of 50 nm bead bound RS218 E. coli.

FIG. 7 is a bar graph showing depletion efficiency of S. aureus usingFcMBL-coated magnetic particles captured by enhanced magnetic separationin the presence of ferromagnetic iron powder that has been treated withdifferent blocking agents.

DETAILED DESCRIPTION OF THE INVENTION

While smaller magnetic particles are more efficient in binding a widerrange of target molecules, methods for effective removal of smallmagnetic particles from a fluid are lacking due to their low magneticmoments. Aspects described herein stem from, at least in part, discoverythat forming a 2D or 3D micro- or nano-structure of magnetic fieldconcentrating particles on a magnetic capture surface or a fluid-contactsurface of a magnetic separation chamber, prior to introducing a fluidsample (comprising magnetic particles, e.g., target-binding magneticparticles) into the chamber to undergo magnetic separation,significantly enhances magnetic separation efficiency of the magneticparticles (e.g., target-binding magnetic particles). When a magneticfield gradient is applied across a magnetic capture surface or magneticseparation chamber, the presence of a 2D or 3D micro- or nano-structureof the magnetic field concentrating particle increases or concentratesthe local magnetic flux density gradient that is experienced by magneticparticles (e.g., target-binding magnetic particles) in a fluid. Thus,magnetic particles (e.g., target-binding magnetic particles) are morereadily attracted to the magnetic field concentrating particles in thepresence of a magnetic field gradient, even when the magnetic moments ofthe target-binding magnetic particles are too low to be removed by theexisting magnetic separation methods. In particular, the inventors havedemonstrated inter alia that the magnetic separation efficiency ofmicrobes (e.g., S. aureus) bound to small magnetic beads (e.g., 50 nm or128 nm in diameter) increased significantly from 15%-30% to at least 95%or higher, when the fluid-contact surface of a microfluidic devicechannel was dispersed with ferromagnetic particles forming a 2D or 3Dmicro- or nano-ferromagnetic structure thereon, prior to introducing afluid to be cleansed for magnetic separation. Additionally, theinventors have used such method to effectively remove pathogeniccontaminants from cord blood.

While the inventors demonstrated the magnetic separation efficiency ofremoving target species (e.g., microbes) from a fluid in a channel,e.g., of a microfluidic device, the concept of forming a 2D or 3D micro-or nano-structure of magnetic field concentrating particles on afluid-contact surface can be extended to magnetic separation of anytarget species using appropriate target-binding magnetic particles in awide range of separation device formats, e.g., non-fluidic and fluidicdevices or systems. Thus, the inventors have developed a novel,versatile and cost-effective method for increasing the magnetic fluxdensity gradient in a magnetic particle-based separation device of anyformat (e.g., tube, multi-well plate, and/or microfluidic channels), andhence improving magnetic separation efficiency of a target molecule froma fluid. Accordingly, aspects described herein relate to methods, kits,devices, and compositions for sensitive magnetic separation or captureof at least one or a plurality of (e.g., at least two or more) targetmolecules from a fluid. The methods, kits, devices, and compositionsdescribed herein can be used for various applications includingcleansing biological fluids as well as food, water, culture medium(e.g., for pharmaceutical manufacturing or brewing), or any other liquidthat can be introduced through a fluidic device.

Methods of Capturing, Separating, or Removing a Magnetic Particle and/ora Target Molecule from a Fluid

One aspect described herein relates to a method of separating magneticparticles from a fluid. The method comprises: (a) subjecting a magneticcapture surface and magnetic field gradient concentrating particles to amagnetic field gradient (a gradient of a magnetic field), wherein themagnetic field gradient concentrating particles, in the presence of themagnetic field gradient, distribute on at least a portion of a magneticcapture surface and substantially align along magnetic flux lines of themagnetic field; and (b) contacting the magnetic capture surface with afluid comprising magnetic particles, wherein the magnetic field gradientconcentrating particles act as local magnetic field gradientconcentrators. At least a portion of the magnetic particles areattracted to the magnetic field gradient concentrating particles in thepresence of the magnetic field gradient, thereby separating the magneticparticles from the fluid. Due to enhancement of the magnetic fieldgradient by magnetic field gradient concentrating particlessubstantially aligning along with magnetic flux lines of a magneticfield applied to the method, such method is particularly useful forseparation of small magnetic particles with a magnetic moment that istoo low to be removed by the existing magnetic separation methods.

As used herein, the term “magnetic capture surface” refers to afluid-contact surface that can provide or generate a magnetic fieldgradient, and/or that can be exposed to a magnetic field gradient duringoperation of magnetic separation. The magnetic capture surface can formpart of a magnetic separation chamber. In some embodiments, the magneticcapture surface can be integral to the magnetic separation chamber. Forexample, the magnetic capture surface can be a surface of a channel(e.g., in a microfluidic device), or a surface of a microwell (e.g., ina multi-well plate). In some embodiments, the magnetic capture surfacecan be detachable from the magnetic separation chamber. For example, asshown in FIG. 6A, the magnetic capture surface 601 is exposed to amagnetic field gradient (e.g., generated by a magnet 603), and magneticfield gradient concentrating particles (e.g., ferromagnetic particles)605 are attracted to the magnetic capture surface 601 in the presence ofthe magnetic field gradient and deposit (e.g., as a layer or anaggregate structure) on at least a portion of the magnetic capturesurface 601. The composite structure comprising the magnetic capturesurface 601 and the magnetic field gradient concentrating particles(e.g., ferromagnetic particles) can then be brought into contact with afluid sample contained in a sample chamber, thereby forming a magneticseparation chamber 607. Inside the magnetic separation chamber 607,magnetic particles (e.g., target-binding magnetic particles) present inthe fluid sample experience a magnetic force due to the magnetic fieldgradient enhanced locally by the magnetic field gradient concentratingparticles 605 and are attracted and bound to the magnetic field gradientconcentrating particles 605. Thus, the magnetic particles (e.g.,target-binding magnetic particles) are separated from the fluid sample.

Due to enhancement of local magnetic field gradients by the presence ofmagnetic field gradient concentrating particles aligning along withmagnetic flux lines of an applied magnetic field, such technique isparticularly useful for separation of small magnetic particles with amagnetic moment that is too low to be removed by the existing magneticseparation methods.

When the magnetic particles are functionalized to specifically bind atarget (e.g., the surface of a magnetic particle is functionalized orcoated with a target-binding molecule on its), the functionalizedmagnetic particles (also referred to herein as “target-binding magneticparticles” can be added to a fluid sample for capture or separation of atarget species, if present, from the fluid. Accordingly, in anotheraspect, described herein is a method of capturing, removing, orseparating a target molecule or species from a fluid or a fluid samplebased on magnetic field gradient concentrating particles dispersed ordistributed on a fluid-contact magnetic capture surface to enhancemagnetic separation strength. The method comprises: introducing a fluidand target-binding magnetic particles to a magnetic separation chambercomprising a magnetic field gradient (a gradient of a magnetic field)therein, wherein at least a portion of a fluid-contact surface of themagnetic separation chamber comprises magnetic field gradientconcentrating particles distributed on the fluid-contact surface. Atleast a portion of the target-binding magnetic particles are attractedto the magnetic field gradient concentrating particles in the presenceof the magnetic field gradient. Thus, a target bound on thetarget-binding magnetic particles is captured, removed, or separatedfrom the fluid.

In some embodiments of this aspect and other aspects described herein,the target-binding magnetic particles can be added to the fluid or fluidsample, prior to introducing the mixture to the magnetic separationchamber comprising a magnetic field gradient therein. Thus, thetarget(s) are allowed to bind to the target-binding magnetic particles,prior to exposing the mixture to a magnetic field gradient for magneticseparation.

In some embodiments of this aspect and other aspects described herein,the fluid or fluid sample and the target-binding magnetic particles canbe added to a sample chamber or an open-top magnetic separation chamberwithout any magnetic field gradient therein. A structure comprising afluid-contact magnetic capture surface and magnetic field gradientconcentrating particles distributed on thereon can then be introducedinto the sample chamber or the open-top magnetic separation chamber sothat the fluid-contact magnetic capture surface is contacted with themixture comprising the fluid and the target-binding magnetic particlescontained in the sample chamber. The magnetic field gradientconcentrating particles distributed on the fluid-contact magneticcapture surface are substantially aligned along magnetic flux lines of amagnetic field (e.g., generated within the structure or appliedexternally to the structure).

In some embodiments of this aspect and other aspects described herein,at least 70% or more (including, e.g., at least 80%, at least 90%, atleast 95%, at least 97%, or more) of the target-binding magneticparticles in a fluid or fluid sample can be captured, separated, orremoved from the fluid or fluid sample.

As used herein, the term “fluid-contact surface” refers to a surface orportion thereof that will be in contact with a fluid upon introductionof the fluid. In some embodiments, the fluid-contact surface is alsosubjected to exposure of a magnetic field gradient for attractingtarget-binding magnetic particles to bind thereon. In some embodiments,the term “fluid-contract surface” also encompasses a magnetic capturesurface.

As used interchangeably herein, the terms “a magnetic field gradient”and “a gradient of a magnetic field” refer to a variation in themagnetic field with respect to position. By way of example only, aone-dimensional magnetic field gradient is a variation in the magneticfield with respect to one direction, while a two-dimensional magneticfield gradient is a variation in the magnetic field with respect to twodirections. The magnetic field gradient can be static or transient. Insome embodiments, the magnetic field gradient can be uniform. In someembodiments, the magnetic field gradient can be non-uniform. Themagnetic field gradient can be generated across a magnetic capturesurface or inside a magnetic separation chamber by any methods known inthe art, e.g., using a magnet, such as a permanent magnet.

In some embodiments of this aspect and other aspects described herein,at least 5% area or more of the fluid-contact surface or magneticcapture surface (that is exposed to a magnetic field gradient) comprisesthe magnetic field gradient concentrating particles distributed thereon.In general, the magnetic separation efficiency increases with largerarea coverage of the fluid-contact surface or magnetic capture surfaceby the magnetic field gradient concentrating particles. Thus, in someembodiments, at least 10% area, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95% or more of the fluid-contact surface or magneticcapture surface (that is exposed to a magnetic field gradient) comprisethe magnetic field gradient concentrating particles distributed thereon.In one embodiment, 100% of the fluid-contact surface or magnetic capturesurface (that is exposed to a magnetic field gradient) comprises themagnetic field gradient concentrating particles distributed thereon.

In some embodiments, the magnetic field gradient concentrating particlesare distributed on at least a portion of (e.g., at least 5% area ormore) the fluid-contact surface or magnetic capture surface to form alayer. The layer can be uniform or uneven in thickness. The layerthickness can vary depending on a number of factors including, e.g., butnot limited to design of a magnetic separation chamber, placement and/ormanipulation of magnet(s) to distribute magnetic field gradientconcentrating particles on a fluid-contact surface, distribution ofmagnetic flux lines, amount of the magnetic field gradient concentratingparticles being distributed on a fluid-contact surface, area of afluid-contact surface to be covered by magnetic field gradientconcentrating particles, and combinations thereof.

To optimize the amount of the magnetic field gradient concentratingparticles used in a magnetic separation chamber and/or the layerthickness of the magnetic field gradient concentrating particles, onecan determine the magnetic isolation efficiency using different amountof the magnetic field gradient concentrating particles on afluid-contact surface. The magnetic isolation efficiency can increasewith the amount of the magnetic field gradient concentrating particlesused, and become plateau beyond a certain point. Thus, in someembodiments, the optimum amount of the magnetic field gradientconcentrating particles can correspond to a point at or close to thebeginning of the plateau, which reflects the highest magnetic isolationefficiency. In some embodiments, the amount of the magnetic fieldgradient concentrating particles distributed on a fluid-contact surfaceor a magnetic capture surface can range from about 0.01 mg/mm² to about5 mg/mm², about 0.025 mg/mm² to about 3 mg/mm², or about 0.05 mg/mm² toabout 1 mg/mm².

In some embodiments, the magnetic field gradient concentrating particlescan form magnetic micro- or nano-structures on at least a portion of thefluid-contact surface of the magnetic separation chamber in the presenceof a magnetic field gradient. The magnetic micro- or nano-structures canbe two dimensional or three dimensional. The magnetic micro- ornano-structures are formed on the fluid-contact surface or magneticcapture surface that is exposed to a magnetic field gradient. By way ofexample only, FIG. 1D or FIG. 1E shows magnetic field gradientconcentrating particles forming protruding or spiky magnetic micro- ornano-structures on a fluid-contact surface or magnetic capture surface.

The magnetic field gradient concentrating particles are substantiallyaligned along the magnetic flux lines of a magnetic field. As usedherein, the term “substantially aligned” includes perfect alignment aswell as alignment with slight deviation from magnetic flux lines of amagnetic field. Perfect alignment refers to perfect alignment of themagnetic field gradient concentrating particles along the magnetic fluxlines of a magnetic field. In some embodiments, the alignment betweenthe magnetic field gradient concentrating particles and magnetic fluxlines of a magnetic field can have a deviation of less than 45°(including, e.g., less than 40°, less than less than 35°, less than 30°,less than 25°, less than 20°, less than 15°, less than 10°, or lessthan) 5° as compared to perfect alignment. For example, FIG. 1D showsthat the magnetic field gradient concentrating particles are arrangedsuch that they follow along the magnetic flux lines of a magnetic fieldgenerated by magnet(s). Exact magnetic flux lines can be determined bycomputational stimulation. The farther away the magnetic flux lines froma magnetic source, the weaker the magnetic strength to fix a magneticflux density gradient concentrating particle in space. Thus, the size ofthe magnetic micro- or nano-structure can vary with the magneticstrength of a magnetic source. By aligning along with magnetic fluxlines of the magnetic field, the magnetic energy of the magnetic fieldgradient concentrating particles is minimized. In some embodiments, themagnetic field gradient concentrating particles can form a fractalstructure on at least a portion of the fluid-contact surface, whichenables generation of stronger magnetic forces around the magnetic fieldgradient concentrating particles. As used herein, the term “fractalstructure” refers to a structure having a repeating pattern thatdisplays at every scale or at every level. A fractal structure is a typeof ordered structures, as distinguished from random structures, whichare not ordered.

The magnetic field gradient concentrating particles dispersed ordistributed on the fluid-contact surface or magnetic capture surface actas magnetic field gradient concentrators. The “magnetic field gradientconcentrators” increase magnetic flux density gradients locallyexperienced by magnetic particles (e.g., target-binding magneticparticles) in a fluid or a fluid sample by at least about 10% or more,as compared to the magnetic flux density gradients experienced by themagnetic particles (e.g., target-binding magnetic particles) in theabsence of the magnetic field gradient concentrators. As used herein,the term “local” or “locally” refers to the magnetic flux densitygradients in the area or space surrounding or nearby the magnetic fieldgradient concentrating particles as experienced by the magneticparticles or target-binding magnetic particles in a fluid, when theyflow past or are in proximity to the magnetic field gradientconcentrators. The size of the local effect can be determined as afunction of a number of factors, including, e.g., but not limited to theapplied magnetic field strength, and/or the size and/or arrangement ofthe magnetic field gradient concentrating particles. In someembodiments, the increase in the local magnetic flux density gradientsexperienced by the magnetic particles (e.g., target-binding magneticparticles) can be at least about 20% or more, including, e.g., at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95% or more, higher than that in the absence of the magnetic fieldgradient concentrators. In some embodiments, the increase in the localmagnetic flux density gradients experienced by the magnetic particles(e.g., target-binding magnetic particles) can be at least about 1.1-foldor more, including, e.g., at least about 1.2-fold, at least about1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at leastabout 2-fold, at least about 3-fold, at least about 4-fold, at leastabout 5-fold, at least about 10-fold, at least about 20-fold, at leastabout 30-fold, at least about 40-fold, at least about 50-fold, at leastabout 60-fold, at least about 70-fold, at least about 80-fold, at leastabout 90-fold, at least about 100-fold, at least about 150-fold, atleast about 200-fold, at least about 300-fold, at least about 400-fold,at least about 500-fold, at least about 1000-fold or more, higher thanthat in the absence of the magnetic field gradient concentrators. Insome embodiments, the increase in the local magnetic flux densitygradients experienced by the magnetic particles (e.g., target-bindingmagnetic particles) can be about 10-fold to about 200-fold, or about10-fold to about 100-fold, higher than that in the absence of themagnetic field gradient concentrators.

By locally increasing the magnetic flux density or magnetic force aroundthe magnetic particles (e.g., target-binding magnetic particles), theefficiency of separating magnetic particles from a fluid and therebycapturing or removing from a fluid at least one or more target moleculesthat are bound to target-binding magnetic particles is enhanced, evenwhen the magnetic particles with weak magnetic moments (e.g., smallmagnetic nanoparticles) are used. In some embodiments, at least about50% or more, including, e.g., at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about98%, or higher and up to 100% of the target-binding magnetic particlesare attracted and bound to the magnetic field gradient concentratingparticles distributed or dispersed on the fluid-contact surface ormagnetic capture surface. In one embodiment, at least about 50% orhigher of the target-binding magnetic particles (e.g., small magneticnanoparticles) are attracted and bound to the magnetic field gradientconcentrating particles distributed or dispersed on the fluid-contactsurface or magnetic capture surface. In one embodiment, at least about90% or higher of the target-binding magnetic particles (e.g., smallmagnetic nanoparticles) are attracted and bound to the magnetic fieldgradient concentrating particles distributed or dispersed on thefluid-contact surface or magnetic capture surface. In one embodiment, atleast about 95% or higher of the target-binding magnetic particles(e.g., small magnetic nanoparticles) are attracted and bound to themagnetic field gradient concentrating particles distributed or dispersedon the fluid-contact surface or magnetic capture surface.

Therefore, the methods of various aspects described herein increases theefficiency of capturing, separating, or removing one or more targetspecies (e.g., at least one, at least two or more target species) from afluid or fluid sample by at least about 30% (including, e.g., at leastabout 60%, at least about 70%, at least about 80%, at least about 90%)or more, as compared to the efficiency in the absence of the magneticfield concentrating particles. In some embodiments, the efficiency ofcapturing, separating, or removing one or more target species (e.g., atleast one, at least two or more target species) from a fluid or fluidsample can be increased by at least about 1.1-fold (including, e.g., atleast about 1.5-fold, at least about 2-fold, at least about 3-fold, atleast about 4-fold) or more, as compared to the efficiency in theabsence of the magnetic field concentrating particles.

In the presence of a magnetic field gradient, the magnetic fieldgradient concentrating particles disperse and conform to at least aportion of a fluid-contact surface of the magnetic separation chamber ora magnetic capture surface. Therefore, the methods of various aspectsdescribed herein are amenable to a wide range of magnetic separationdevices in various configurations. The magnetic separation devicecomprises a magnetic separation chamber or a magnetic capture surface asdefined herein. As used herein, the term “magnetic separation chamber”refers to a chamber or space subjected to exposure of a magnetic fieldgradient and comprising at least one inlet for introduction of a fluid,and optionally at least one outlet for exit of the fluid. In someembodiments, the inlet can be used as an outlet for exit of a fluid. Themagnetic separation chamber includes a fluid-contact surface that issubjected to exposure of a magnetic field gradient. For example, themagnetic separation chamber can comprise a channel, a microfluidicchannel, a sample well, a microtiter plate, a slide (e.g., a glassslide), a flask (e.g., a tissue culture flask), a tube, a nanotube, afiber, a filter, a membrane, a scaffold, an extracorporeal device, amixer, a hollow fiber, or any combinations thereof. Thus, the magneticseparation chamber can be of any shape and/or any size. In oneembodiment, the fluid-contact surface of the magnetic separation chamberis a surface within a channel or a microchannel. In one embodiment, thefluid-contact surface of the magnetic separation chamber is a surface ofa microwell. In one embodiment, the fluid-contact surface of themagnetic separation chamber is a surface of a magnetic solid substrate(e.g., in a form of a protruding structure such as a tip) that isbrought into contact with a fluid.

In some embodiments, the magnetic separation chamber is a non-fluidicchamber, in which a fluid remains in the chamber and does not flowthrough the chamber. Examples of a non-fluidic chamber include, withoutlimitations, a multi-well plate, a flask (e.g., a tissue culture flask),a tube (e.g., eppendorf tubes, or tubes with an opening), any samplecarriers, and combinations thereof. Thus, in some embodiments, themethods described herein can be applied to non-fluidic magneticseparation applications. By way of example only, the methods describedherein can be used with a commercially available non-fluidic device,e.g., a magnetic column (e.g., Miltenyi®). The magnetic field gradientconcentrating particles can be used in place of steel or iron wool anddispersed on the interior surface of the column in the presence of amagnetic field gradient, followed by adding a fluid sample comprisingtarget-binding magnetic particles described herein. Another example of anon-fluidic chamber for use in the method described herein is amulti-well plate (e.g., 96-well plate), where the magnetic fieldgradient concentrating particles formed 2D or 3D magnetic nano- ormicro-structures on the fluid-contact surface within the wells (e.g., onthe magnetic tips of the movable multi-well tip comb brought into thewells as shown in FIG. 6A, or the well surface experiencing a magneticfield gradient as shown in FIGS. 6B-6C).

In some embodiments, the magnetic separation chamber is a fluidicchamber, which allows a fluid flowing therethrough. Examples of afluidic chamber include, without limitations, a channel, a microfluidicchannel, a hollow fiber, a hollow tube. Thus, in some embodiments, themethod described herein can be applied to fluidic magnetic separationapplications, e.g., as described in the Examples using a microfluidicdevice for magnetic separation. See, e.g., WO/2011/091037,WO/2012/135834, and WO 2013/126774, the contents of which areincorporated herein by reference, for additional microfluidic devicesthat can be used with the method described herein.

In some embodiments where the magnetic separation chamber is a fluidicchamber, the fluid can be introduced to the chamber at any flow rate,for example, depending on the volume capacity of the magnetic separationchamber, magnetic properties of the magnetic field gradientconcentrating particles and/or target-binding magnetic particles, and/orthe magnetic field gradient. In some embodiments, the fluid can beintroduced to the chamber at a flow rate of about 0.1 mL/hr to about 100L/hr. In some embodiments, the fluid can be introduced to the chamber ata flow rate of about 0.5 mL/hr to about 50 L/hr. In some embodiments,the fluid can be introduced to the chamber at a flow rate of about 1mL/hr to about 10 L/hr. In some embodiments, the fluid can be introducedto the chamber at a flow rate of about 50 mL/hr to about 10 L/hr. Insome embodiments, the fluid can be introduced to the chamber at a flowrate of about 1 L/hr to about 100 L/hr. In some embodiments, the fluidcan be introduced to the chamber at a flow rate of about 1 L/hr to about50 L/hr. In some embodiments, the fluid can be introduced to the chamberat a flow rate of about 50 mL/hr to about 10 L/hr.

In accordance with the methods of various aspects described herein, themagnetic field gradient concentrating particles are dispersed ordistributed onto the fluid-contact surface or magnetic capture surface,prior to contacting the fluid-contact surface or magnetic capturesurface with a fluid or a fluid sample comprising the target-bindingmagnetic particles. Thus, the fluid to be brought into contact with thefluid-contact surface or magnetic capture surface can comprise thetarget-binding magnetic particles but does not contain magnetic fieldgradient concentrating particles suspended in the same fluid. One of theadvantages of the method described herein is that the fluid can beintroduced into the chamber at high flow rates, enabling ahigh-throughput separation process. On the other hand, if the magneticfield gradient concentrating particles and target-binding magneticparticles were to be suspended in the same fluid during magneticcapture, the magnetic separation efficiency would be low at high flowrates. It is because in the presence of a magnetic field gradient, themagnetic field gradient concentrating particles and target-bindingmagnetic particles could migrate differently in the fluid, for example,due to a difference in magnetic drag velocity. By way of example only,when a fluid containing larger magnetic field gradient concentratingparticles (e.g., ˜1 μm in diameter) and smaller target-binding magneticparticles (e.g., ˜128 nm) were to be flowed at high flow rates (e.g.,˜100 mL/s to 1000 mL/s) in the presence of a magnetic field gradient,the larger magnetic field gradient concentrating particles would havemuch faster magnetic drag velocity and thus be attracted to a surfaceand pulled away from the target-binding magnetic particles. Therefore,the magnetic field gradient concentrating particles were not able to actas magnetic field gradient concentrators for the target-binding magneticparticles.

In one embodiment, prior to contacting the fluid-contact surface ormagnetic capture surface with the fluid comprising target-bindingmagnetic particles, the method can further comprise distributing ordispersing the magnetic field gradient concentrating particles onto atleast a fluid-contact surface or magnetic capture surface. The magneticfield gradient concentrating particles conform to the fluid-contactsurface or magnetic capture surface. To do this, for example, themagnetic field gradient concentrating particles suspended in a buffer oran organic solvent (e.g., ethanol) can be introduced into a magneticseparation chamber while a magnetic field gradient is present to attractor trap the magnetic field gradient concentrating particles onto atleast a portion of the fluid-contact surface of the magnetic separationchamber. Methods to produce a magnetic field gradient to attractmagnetic particles are known in the art. Once the magnetic fieldgradient concentrating particles are trapped in the magnetic separationchamber or trapped on a magnetic capture surface, the magnetic fieldgradient can be manipulated to distribute or disperse those magneticfield gradient concentrating particles over the fluid-contact surfacearea in a desired manner (e.g., uniform distribution, randomdistribution, or in a specific pattern). The magnetic field gradientconcentrating particles are magnetized and substantially aligned alongthe magnetic flux lines of a magnetic field. In one embodiment, at leastone or more permanent magnets can be placed around the magneticseparation chamber or be placed on at least one side of a magneticcapture surface and be manipulated to distribute or disperse thosemagnetic field gradient concentrating particles over the fluid-contactsurface area in a desired manner (e.g., uniform distribution, randomdistribution, or in a specific pattern).

In some embodiments, the method can comprise collecting target-boundtarget-binding magnetic particles, for example, for subsequent analysisor analyses, including, e.g., but not limited to, ELISA, opticalimaging, spectroscopy, polymerase chain reaction (PCR), massspectrometric methods, immunoassays, and any combinations thereof. Priorto the analysis, the target-bound target-binding magnetic particles canbe released from the magnetic field gradient concentrating particles,for example, by fluid shear stress, or demagnetizing the magnetic fieldgradient concentrating particles and/or target-bound target-bindingmagnetic particles.

In some embodiments, the method can comprise subjecting the fluid, uponmagnetic capture of the target-bound target-binding magnetic particles,to at least a detection assay or analysis to determine if a targetmolecule has been captured or removed by the target-binding magneticparticles.

By having one or a plurality of (e.g., at least 2, at least 3 or more)different target-binding magnetic particles in a fluid, the methoddescribed herein can be adapted to capture or remove or capture at leastone type of target molecules or a plurality of (e.g., at least 2, atleast or more) different types of target molecules from the fluid. Insome embodiments, different target-binding magnetic particles can beadded to the fluid all at once and the fluid is then subjected tomagnetic separation using the method as described herein to remove orcapture various target molecules. By varying at least one or moreparameters of magnetic separation, including, e.g., flow rate of thefluid, the strength and/or duration of the magnetic field gradient,and/or the magnetic properties and/or size of the target-bindingmagnetic particles and/or magnetic field gradient concentratingparticles, different types of target-bound target-binding magneticparticles can be selectively attracted to the magnetic field gradientconcentrating particles that are distributed on the fluid-contactsurface. By way of example only, where target-binding magnetic particlesof different sizes are used to bind distinct target molecules, differentsized target-binding magnetic particles can be pulled to a fluid-contactsurface (comprising the magnetic field gradient concentrating particlesdistributed thereon) at different rates. In a fluidic magneticseparation device, different sized target-binding magnetic particles(with the corresponding target molecules bound thereto) can be fluiddynamically aligned in a certain portion of a fluidic channel prior tomagnetic separation. Fluid dynamically alignment is based on a principlecalled inertia hydrodynamic focusing that has been used for sheathlessfocusing of particles in channels. As a fluid flows through the magneticseparation chamber in the presence of a magnetic field gradient, thetarget-binding magnetic particles of different sizes in the fluid can bepulled to different portions of a fluid-contact surface of the magneticseparation chamber. Accordingly, in these embodiments, different typesof target molecules can be selectively captured or removed from thefluid using target-binding magnetic particles of different sizes. Assuch, in some embodiments, the methods described herein can comprise,prior to magnetic separation, pre-aligning the target-binding magneticparticles of different sizes within a fluidic channel such that theywill flow from the same starting line within the fluidic channel whenthey are exposed to a magnetic field gradient. Depending on the size ofthe magnetic particles and/or the magnetic force acting on them, thetarget-binding magnetic particles of different sizes can be selectivelypulled to different portions of the fluid-contact surface comprisingmagnetic field gradient concentrating particles distributed thereon. Thebound target-binding magnetic particles can be collected afterward.

In alternative embodiments, different target molecules can beindividually captured or removed from the fluid in a sequential manner.For example, a first type of target molecules can be first captured orremoved by adding a first type of target-binding magnetic particles tothe fluid for magnetic separation using the method as described herein.Capture or removal of a second type of target molecules from the fluidcan follow by repeating the method described herein with a second typeof target-binding magnetic particles.

The methods of various aspects described herein can be utilized tocapture or remove any target molecules of interest in any fluid.Non-limiting examples of the target molecules that can be captured orremoved using the method described herein include cells, proteins,nucleic acids, microbes, small molecules, chemicals, toxins, drugs, andany combinations thereof. In one embodiment, the method described hereinis adapted to capture or remove a microbe from a fluid.

The magnetic field source used in the methods of various aspectsdescribed herein can be any magnet device that can be positioned togenerate a magnetic field gradient for trapping magnetic field gradientconcentrating particles on a fluid-contact surface or a magnetic capturesurface and thereby increasing local magnetic field gradient experiencedby a magnetic particle (e.g., a target-binding magnetic particle). Anelectromagnetic controller can be used to control and adjust themagnetic field and gradients thereof, and to control the distribution ofthe magnetic field gradient concentrating particles on a fluid-contactsurface or a magnetic capture surface. The magnetic field gradient canbe generated by a permanent magnet or by an electromagnetic signalgenerator. The electromagnetic signal generator can include anelectromagnet or electrically-polarizable element, or at least onepermanent magnet. The magnetic field gradient can be produced at leastin part according to a pre-programmed pattern. The magnetic fieldgradient can have a defined magnetic field strength and/or spatialorientation. In some embodiments, the magnetic field gradient has adefined magnetic field strength.

As used herein, the term “magnetic field” refers to magnetic influenceswhich create a local magnetic flux that flows through a composition andcan refer to field amplitude, squared-amplitude, or time-averagedsquared-amplitude. It is to be understood that magnetic field can be adirect-current (DC) magnetic field or alternating-current (AC) magneticfield. The magnetic field strength can range from about 0.00001 Teslaper meter (T/m) to about 10⁵ T/m. In some embodiments, the magneticfield strength can range from about 0.0001 T/m to about 10⁴ T/m. In someother embodiments, the magnetic field strength can range from about0.001 T/m to about 10³ T/m.

Microbe capture from a fluid: In some embodiments, the methods ofvarious aspects described herein can be used to capture or remove atleast one or more microbes from a fluid. In some embodiments, themethods described herein can be used in combination with any methods forcapturing or removing microbe(s) from a fluid using microbe-bindingmagnetic particles described in WO/2011/090954 and WO/2013/012924,contents of both of which are incorporated herein by reference in theirentireties. For example, the fluid-contact surface of the microbecapture devices (e.g., comprising a non-fluidic or fluidic magneticseparation chamber as described herein) can be distributed or dispersedwith magnetic field gradient concentrating particles to form magneticnano- or micro-structures, prior to introduction of a fluid having orsuspected of having microbe(s).

In some embodiments, the methods described herein can be performed in amicrobe diagnostic device or blood cleansing device as described in Int.Pat. App. No. WO 2011/091037, filed Jan. 19, 2011, and/or WO 2012/135834filed Apr. 2, 2012, the contents of which are incorporated herein byreference in their entireties. In these embodiments, the fluid-contactsurface of the microbe diagnostic device or blood cleansing device canbe distributed or dispersed with magnetic field gradient concentratingparticles to form magnetic nano- or micro-structures, prior tointroduction of a fluid having or suspected of having microbe(s). Amicrobe diagnostic device as described in Int. Pat. App. No. WO2011/091037, filed Jan. 19, 2011, can also be modified to replace thecapture chamber or capture and visualization chamber with an s-shapedflow path. A magnet can then be used to capture bound microbe againstthe flow path wall; separating the bound microbe from rest of the fluid.

In some embodiments, the methods described herein can be performed in adevice and/or in combination with a method as described in U.S. Pat.App. Pub. No. 2009/0220932, No. 2009/007861, No. 2010/0044232, No.2007/0184463, No. 2004/0018611, No. 2008/0056949, No. 2008/0014576, No.2007/0031819, No. 2008/0108120, and No. 2010/0323342, the contents ofwhich are all incorporated herein by reference.

Magnetic Field Gradient Concentrating Particles

As described earlier, the magnetic field gradient concentratingparticles are magnetic particles that act as local magnetic fieldgradient concentrators to increase the local magnetic flux densitygradient experienced by a magnetic particle (e.g., a target-bindingmagnetic particle) in a magnetic separation chamber and hence increaseefficiency of separating or capturing target-binding magnetic particlesfrom a fluid. The term “magnetic field gradient” as used herein refersto a variation in the magnetic field with respect to position. By way ofexample only, a one-dimensional magnetic field gradient is a variationin the magnetic field with respect to one direction, while atwo-dimensional magnetic field gradient is a variation in the magneticfield with respect to two directions. The magnetic field gradient can bestatic or transient (dynamic). For example, a transient magnetic fieldgradient can be a rotating or translational magnetic field gradient.

Magnetic field gradient concentrating particles can be commerciallyavailable magnetic particles of desired sizes. Magnetic particles(including nanoparticles or microparticles) are well-known and methodsfor their preparation have been described in the art. See, e.g., U.S.Pat. Nos. 6,878,445; 5,543,158; 5,578,325; 6,676,729; 6,045,925; and7,462,446; and U.S. Patent Publications No. 2005/0025971; No.2005/0200438; No. 2005/0201941; No. 2005/0271745; No. 2006/0228551; No.2006/0233712; No. 2007/01666232; and No. 2007/0264199.

The magnetic field gradient concentrating particles can comprisesuperparamagnetic particles, paramagnetic particles, ferrimagneticparticles, ferromagnetic particles, or combinations thereof. The term“paramagnetic” as used herein means a material with a small but positivemagnetic susceptibility (magnetigability). Paramagnetic particles areattracted by an external magnetic field, and form internal, inducedmagnetic fields in the direction of the applied magnetic field. However,paramagnetic materials do not retain any magnetization in the absence ofan external magnetic field. Examples of paramagnetic materials that canbe included in the magnetic field gradient concentrating particlesinclude, without limitations, metal ions (e.g., Gd3+, Fe3+, Mn2+, andCu2+), transition metals, such as titanium, vanadium, chromium,manganese, iron cobalt, nickel, copper, and compounds thereof;lanthanide metals, such as europium and gadolinium, and compoundsthereof; rare earth elements and compounds thereof; free radicals, suchas nitroxides and compounds thereof; and actinide metals, such asprotactinium, and compounds thereof.

As used herein, the term “ferromagnetic” refers to a substance, such asiron particles, having a large positive magnetic susceptibility. Theseparticles possess a large magnetic moment and are able to relaxneighboring nuclei much faster than paramagnetic ions. They possesslarge magnetic moments even in weak external fields and produce largelocal magnetic flux densities. Unlike paramagnetic materials,ferromagnetic materials retain magnetization even when the externalmagnetic field gradient is removed. Ferromagnetic materials that can beincluded in the magnetic field gradient concentrating particles include,but are not limited to, iron oxides, such as Fe₂O₃, reduced iron, ironpowder, atomized iron, electrolyte iron, cobalt, nickel, permalloy,alloys comprising at least one or more ferromagnetic materials describedherein, some compounds of rare earth metals, some minerals such asiodestone, and a combination of two or more thereof. There are a numberof methods to manufacture iron particles. Reduced iron is also calledsponge iron because it contains sponge-like hollow spaces inside theparticles. Atomization of iron (atomized iron) can be made by forcing amolten metal stream (e.g., a molten stream comprising iron) through anarrow duct at high pressure.

In some embodiments, magnetite particles are ferromagnetic when theirsizes are above a certain threshold, e.g., 10 nm. When the magnetiteparticle sizes are below 10 nm, the magnetized vector becomes unstable,and the magnetic property is no longer ferromagnetic but“superparamagnetic.”

In one embodiment, the magnetic field gradient concentrating particlesare ferromagnetic particles. In one embodiment, the ferromagneticparticles comprise atomized iron. Ferromagnetic materials or particlesgenerally exhibit a larger magnetic permeability than paramagneticmaterials, and are thus able to keep magnetic fields within themselveswhen they are exposed to an external magnetic field. In the absence ofan external magnetic field, the magnetic domains of ferromagneticmaterials stay randomly oriented due to thermal fluctuations. However,they become substantially aligned parallel under the applied magneticfields. The magnetic fields converge into ferromagnetic materials orparticles due to their high magnetic permeability to reduce the magneticenergy, and the ferromagnetic materials or particles then generate highmagnetic flux density gradient by having magnetic fields diverge intothe surrounding free space, thus providing stronger magnetic forces.

By the term “superparamagnetic” is meant a material that is highlymagnetically susceptible, i.e., it becomes strongly magnetic when placedin a magnetic field, like in ferromagnetism; however, like aparamagnetic material, a superparamagnetic material rapidly loses itsmagnetism and displays no remanence once the magnetic field is removed.

As used herein, the term “ferrimagnetic” includes materials that havepopulations of atoms with opposing magnetic moments, as inantiferromagnetism; however, in ferrimagnetic materials, the opposingmoments are unequal and a spontaneous magnetization remains. Examples offerrimagnetic materials that can be included in the magnetic fieldgradient concentrating particles include, but are not limited toferrites, magnetic garnets, magnetite (iron (II, III) oxide), yttriumiron garnet, cubic ferrites comprising iron oxides and other elementssuch as aluminum, cobalt, nickel, manganese and zinc, hexagonal ferritessuch as PbFe₁₂O₁₉ and BaFe₁₂O₁₉, and pyrrhotite, Fe_(1-X)S.

The magnetic field gradient concentrating particles can be selected forany size, e.g., depending on the dimensions of the magnetic separationchamber and/or area of the fluid-contact surface. For example, the sizeof the magnetic field gradient concentrating particles can be smallerthan (e.g., by at least 10% or more), comparable to (e.g., within 10%,or within 5%), or larger than (e.g., by at least 10% or more) that ofthe target-binding magnetic particles described herein. In someembodiments, the magnetic field gradient concentrating particles arelarger than the target-binding magnetic particles. In some embodiments,the size (e.g., diameter) of the magnetic field gradient concentratingparticles are larger than the size (e.g., diameter) of thetarget-binding magnetic particles by at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about98%, or more. In some embodiments, the size (e.g., diameter) of themagnetic field gradient concentrating particles are larger than the sizeof the target-binding magnetic particles by at least about 1-fold, atleast about 2-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 6-fold, at least about 7-fold, atleast about 8-fold, at least about 9-fold, at least about 10-fold, atleast about 15-fold, at least about 20-fold, at least about 50-fold, atleast about 100-fold, at least about 1000-fold or higher.

In some embodiments, the magnetic field gradient concentrating particlesare smaller than the target-binding magnetic particles. In someembodiments, the size (e.g., diameter) of the magnetic field gradientconcentrating particles are smaller than the size (e.g., diameter) ofthe target-binding magnetic particles by at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, or more. When the magnetic fieldgradient concentrating particles are smaller than the target-bindingmagnetic particles in size, the magnetic field gradient concentratingparticles can aggregate and form magnetic micro- or nano-structures oncethey are exposed to an applied magnetic field.

In some embodiments, the size (e.g., diameter) of the magnetic fieldgradient concentrating particles ranges from about 50 nm to about 5 mm,from about 100 nm to about 3 mm, from about 1 μm to about 1 mm. In oneembodiment, the diameter of the magnetic field gradient concentratingparticles is about 300 μm.

The magnetic field gradient concentrating particles can be of any shape,including but not limited to spherical, rod, elliptical, cylindrical,and disc. In some embodiments, magnetic field gradient concentratingparticles having a substantially spherical shape and defined surfacechemistry can be used to minimize chemical agglutination andnon-specific binding.

In some embodiments, the magnetic field gradient concentrating particlescan be functionalized or unfunctionalized. In some embodiments, themagnetic field gradient concentrating particles can be functionalized(e.g., by conjugating a chemical functional group or a molecule to themagnetic field gradient concentrating particles). By way of exampleonly, in some embodiments, the magnetic field gradient concentratingparticles can be functionalized with molecules that allow target-bindingmagnetic particles to remain bound to the magnetic field gradientconcentrating particles in the absence of a magnetic field.Target-binding magnetic particles are attracted to the magnetic fieldgradient concentrating particles when a magnetic field is applied. Afterthe magnetic field is removed, the magnetic attraction between themagnetic field gradient concentrating particles and the target-bindingmagnetic particles can decrease over time, or become insufficient towithstand external shear stress introduced during an assay.Functionalization of the magnetic field gradient concentrating particlescan inhibit or minimize the likelihood of target-binding magneticparticles that were attracted to the magnetic field gradientconcentrating particles to release from the surface of the magneticfield gradient concentrating particles, when a magnetic field isremoved, and/or during the course of an assay, e.g., various washprocedures of ELISA or immunoassays. This can help increasing theaccuracy and/or sensitivity of an assay for detection of targetmolecules in a fluid.

In some embodiments, the magnetic field gradient concentrating particlescan be unfunctionalized, e.g., magnetic particles with no activefunctional groups on their surfaces for conjugation or any otherreaction with the surrounding.

In some embodiments, the magnetic field gradient concentrating particlescan be modified to inhibit non-specific binding of target molecules tothe magnetic field gradient concentrating particles.

In some embodiments of this aspect and other aspects described herein,the magnetic field gradient concentrating particles by themselves arenot able to bind or capture a target. In some embodiments of this aspectand other aspects described herein, the magnetic field gradientconcentrating particles do not comprise metal oxide (e.g., iron oxide).In some embodiments, the magnetic field gradient concentrating particlescan be treated to reduce non-specific interaction, for example, by atleast about 30% or more (including, e.g., at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95% or more), with a target to beremoved or separated from a fluid. This can improve selectivity of themethods, kits, or solid substrates described herein to capture, remove,or separate target molecule(s) from a fluid. For example, in someembodiments, the magnetic field gradient concentrating particles can betreated with a blocking agent, e.g., to reduce their non-specificinteraction with a target to be removed or separated from a fluid.Non-limiting examples of a blocking agent include a lubricant (e.g., butnot limited to silicone, or mold-release agent), a polymer (e.g., butnot limited to silicon-based polymer such as polydimethylsiloxane(PDMS)), milk proteins, bovine serum albumin, blood serum, whole blood,and a combination of two or more thereof.

In some embodiments of this aspect and other aspects described herein,the magnetic field gradient concentrating particles by themselves canbind or capture a target molecule or species to be captured, removed, orseparated from a fluid. In some embodiments, the magnetic field gradientconcentrating particles can be adapted or modified to bind or capture atarget molecule or species to be captured, removed, or separated from afluid. In some embodiments where the magnetic field gradientconcentrating particles have target-binding capability, using bothtarget-binding magnetic particles and magnetic field gradientconcentrating particles in the methods, devices, kits, and/or solidsubstrates described herein can produce a synergistic effect incapturing, removing, or separating a target molecule or species from afluid. In some embodiments where the magnetic field gradientconcentrating particles have target-binding capability, using bothtarget-binding magnetic particles and magnetic field gradientconcentrating particles in the methods, devices, kits, and/or solidsubstrates described herein can produce an additive effect in capturing,removing, or separating a target molecule or species from a fluid.

Magnetic Particles and Target-Binding Magnetic Particles

As used herein, the term “magnetic particle” refers to a particle thatcan be magnetically attracted to one or more magnetic field gradientconcentrating particles when the magnetic field gradient concentratingparticle(s) is/are subjected to a magnetic field gradient.

As used herein, the term “target-binding magnetic particle” refers to amagnetic particle adapted to specifically bind at least one or moretarget molecules described herein. For example, the exterior surface ofthe magnetic particle comprises target-binding molecules conjugatedthereto. Depending upon applications of the method described herein, thetarget-binding magnetic particles can be adapted to bind one or moretarget molecules in any type of fluid described herein.

As used herein, the term “specifically binds” refers to the ability of atarget-binding magnetic particle or target-binding molecule to bind to aspecific target species or molecule with a K_(D) 10⁻⁵ M (10000 nM) orless, e.g., 10⁻⁶ M or less, 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M orless, 10⁻¹¹ M or less, 10⁻¹¹ M or less, or 10⁻¹² M or less. For example,if a microbe-binding molecule (e.g., MBL or a functional fragmentthereof) binds to a microbe and/or microbe-associated molecular pattern(MAW) with a K_(D) of 10⁻⁵ M or lower, but not substantially tomolecules that are not microbes or MAMPs, then the agent is said tospecifically bind the microbe and/or MAMP. By “not substantially” ismeant that the K_(D) for target molecules, as determined, e.g., bycompetition assay or by other means known in the art, is at least10²-fold lower than that for other non-target molecules, and preferablyat least 10³-fold lower, at least 10⁴-fold lower, 10⁵-fold lower orless. Specific binding can be influenced by, for example, the affinityand avidity of the target-binding molecules and the concentration of thetarget-binding molecules and/or target-binding magnetic particles used.A person of ordinary skill in the art can determine appropriateconditions under which the target-binding magnetic particles describedherein selectively bind using any suitable methods, such as titration oftarget-binding magnetic particles in a suitable assay and depletionefficiency of the target from a sample, such as those described hereinin the Examples.

In some embodiments, at least a portion of the target-binding magneticparticles can have the corresponding target molecules bound thereto. Insome embodiments, at least a portion of the target-binding magneticparticles do not have any target molecules bound thereto.

The target-binding magnetic particles can be paramagnetic,superparamagnetic, or ferromagnetic. In some embodiments, thetarget-binding magnetic particles can be paramagnetic orsuperparamagnetic. In some embodiments, the target-binding magneticparticles can have the same core magnetic particles as the magneticfield gradient concentrating particles, optionally with differentsurface properties, e.g., surface chemistry and/or composition. In otherembodiments, the core magnetic particles within the target-bindingmagnetic particles can be different from that of the magnetic fieldgradient concentrating particles.

In some embodiments, the target-binding magnetic particles can betarget-binding magnetic particles with weak magnetic moments that theycannot be efficiently captured or removed from the fluid in the presenceof an external magnetic field without the magnetic field gradientconcentrating particles described herein or using the existing magneticseparation techniques. Magnetic moment or magnetic dipole moment (M) isproportional to magnetic susceptibility (x) (M=f H, where H is appliedmagnetic field strength). Accordingly, in some embodiments, thetarget-binding magnetic particles can be target-binding magneticparticles with magnetic susceptibility ranging from less than 1 emu/g toabout 100 emu/g. In some embodiments, the target-binding magneticparticles can be target-binding magnetic particles with magneticsusceptibility of about less than 1 emu/g to about 80 emu/g, or about 5emu/g to about 75 emu/g. In some embodiments, the target-bindingmagnetic particles can be target-binding magnetic particles withmagnetic susceptibility of about 40 emu/g to about 50 emu/g. Forexample, iron oxide nano- or micro-particles smaller than 4 μm can havean increased magnetic separation efficiency when they work with themagnetic field gradient concentration particles described herein.

While any size (e.g., ranging from about 1 nm to about 1 mm) of thetarget-binding magnetic particles can be used in the method describedherein, the methods of various aspects described herein can provide aneffective solution to magnetic separation with small target-bindingmagnetic particles (or magnetic particles with weak magnetic moments),because the existing magnetic separation methods generally do not workor yield a low magnetic separation efficiency with small target-bindingmagnetic particles due to their weak magnetic moments. Accordingly, insome embodiments, the target-binding magnetic particles arenanoparticles. As used herein, the term “nanoparticle” refers toparticles that are on the order of 10⁻⁹ or one billionth of a meter andbelow. Generally, nanoparticles have a diameter in the range from about1 nm to about 1000 nm. The term “nanoparticle” includes e.g., but is notlimited to, nanospheres; nanorods; nanoshells; and nanoprisms. In someembodiments, the diameter of the target-binding magnetic nanoparticlesis no more than 250 nm, no more than 100 nm, no more than 50 nm, or nomore than 5 nm.

In some embodiments, the target-binding magnetic particles aremicroparticles smaller than 10 μm, including, e.g., smaller than 5 μm,smaller than 4 μm, smaller than 3 μm, smaller than 2 μm, smaller than 1μm or less.

In some embodiments, target-binding magnetic particles can comprise ontheir surfaces target-binding molecules. By “target-binding molecules”is meant herein molecules that can interact with or bind to a targetspecies or a target molecule of interest such that the target species ortarget molecule can be captured or removed from a fluid. Typically thenature of the interaction or binding is noncovalent, e.g., by hydrogen,electrostatic, or van der Waals interactions, however, binding can alsobe covalent. Target-binding molecules can be naturally-occurring,recombinant or synthetic. Examples of the target-binding molecule caninclude, but are not limited to, a nucleic acid, an antibody or aportion thereof, an antibody-like molecule, an enzyme, an antigen, asmall molecule, a protein, a peptide, a peptidomimetic, a carbohydrate,an aptamer, and any combinations thereof. By way of example only, forremoval of microbes from a fluid, the target-binding molecule can beselected from the group consisting of: opsonins, lectins, antibodies,and antigen binding fragments thereof, proteins, peptides,peptidomimetics, carbohydrate-binding proteins, nucleic acids,carbohydrate, lipids, steroids, hormones, lipid binding molecules,cofactors, nucleosides, nucleotides, peptidoglycan,lipopolysaccharide-binding proteins, small molecules, and anycombinations thereof. An ordinary artisan can readily identifyappropriate target-binding molecules for each target species or targetmolecules of interest to be captured or removed from a fluid.

In some embodiments, the target-binding molecules can be modified by anymeans known to one of ordinary skill in the art. Methods to modify eachtype of target-binding molecules are well recognized in the art.Depending on the types of target-binding molecules, an exemplarymodification includes, but is not limited to genetic modification,biotinylation, labeling (for detection purposes), chemical modification(e.g., to produce derivatives or fragments of the target-bindingmolecule), and any combinations thereof. In some embodiments, thetarget-binding molecule can be genetically modified. In someembodiments, the target-binding molecule can be biotinylated.

In some embodiments, the target-binding molecules can comprise on theirsurfaces microbe-binding molecules as described herein, and/or disclosedin WO/2011/090954 and WO/2013/012924, the contents of which areincorporated herein by reference. Accordingly, in some embodiments, themethod described herein can be used with the target-binding magneticparticles for microbial capture, i.e., microbe-binding magneticparticles, e.g., but not limited to FcMBL magnetic particles. Examplesof microbe-binding magnetic particles can include, but are not limitedto the ones described in WO/2011/090954 and WO/2013/012924, the contentsof which are incorporated herein by reference.

In some embodiments, the target-binding molecule can be an antibody or aportion thereof, or an antibody-like molecule. In some embodiments, thetarget-binding molecule can be an antibody or a portion thereof, or anantibody-like molecule that is specific for detection of a rare-cell,e.g., a circulating tumor cell, a fetal cell, a stem cell and/or amicrobe biomarker. In some embodiments, the target-binding molecule canbe an antibody or a portion thereof, or an antibody-like molecule thatis specific for a protein or an antigen present on the surface of a rarecell, e.g., a circulating tumor cell, a fetal cell, a stem cell and/or amicrobe. In such embodiments, the target-binding molecules can be usedto, for example, detect and/or identify cell type or species (includingnormal and/or diseased cells), the presence of cell or disease markers,cellular protein expression levels, phosphorylation or otherpost-translation modification state, or any combinations thereof.

In some embodiments, the target-binding molecule can be a nucleic acid(e.g., DNA, RNA, LNA, PNA, or any combinations thereof). For example,the nucleic acid can encode the gene specific for a rare cell biomarker,e.g., a circulating tumor cell, a fetal cell, a stem cell and/or amicrobe biomarker. In such embodiments, the nucleic acids can be used todetermine, for example, the existence of characteristic cellular DNA orRNA sequences (such as in fluorescent in situ hybridization), RNAexpression levels, miRNA presence and expression, and any combinationsthereof, in various applications, e.g., for disease diagnose, prognosisand/or monitoring.

In some embodiments, the target-binding molecule can be a protein or apeptide. In some embodiments, the protein or peptide can be essentiallyany proteins that can bind to a rare cell, e.g., a circulating tumorcell, a fetal cell, a stem cell and/or a microbe. By way of exampleonly, if the target species is a bacteria, exemplary proteins orpeptides that can be used to generate microbe-binding molecules and/ormicrobe-binding magnetic particles can include, but are not limited to,innate-immune proteins (e.g., without limitations, MBL, Dectin-1, TLR2,and TLR4 and any molecules (including recombinant or engineered proteinmolecules) disclosed here as well as the microbe-binding moleculesdisclosed in the International Application Publication Nos.WO/2011/090954 and WO/2013/012924, the content of which is incorporatedherein by reference) and proteins comprising the chitin-binding domain,and any factions thereof. Such innate-immune proteins and chitin-bindingdomain proteins can be used to detect their correspondingpattern-recognition targets (e.g., microbes such as bacteria) andfungus, respectively.

In some embodiments, the target-binding molecule can be an aptamer. Insome embodiments, the target-binding molecule can be a DNA or RNAaptamer. The aptamers can be used in various bioassays, e.g., in thesame way as antibodies or nucleic acids described herein. For example,the DNA or RNA aptamer can encode a nucleic acid sequence correspondingto a rare cell biomarker or a fraction thereof, for use as atarget-binding molecule on the magnetic particles described herein.

In some embodiments, the target-binding molecule can be a cell surfacereceptor ligand. As used herein, a “cell surface receptor ligand” refersto a molecule that can bind to the outer surface of a cell. Exemplarycell surface receptor ligand includes, for example, a cell surfacereceptor binding peptide, a cell surface receptor binding glycopeptide,a cell surface receptor binding protein, a cell surface receptor bindingglycoprotein, a cell surface receptor binding organic compound, and acell surface receptor binding drug. Additional cell surface receptorligands include, but are not limited to, cytokines, growth factors,hormones, antibodies, and angiogenic factors. In some embodiments, anyart-recognized cell surface receptor ligand that can bind to a rarecell, e.g., a circulating tumor cell, a fetal cell, a stem cell and/or amicrobe, can be used as a target-binding molecule on the magneticparticles described herein.

Microbe-Binding Magnetic Particles

In some embodiments, the target-binding magnetic particles are adaptedto specifically bind at least one or more microbes or fragments thereof(referred to as “microbe-binding magnetic particles”) ormicrobe-associated molecular patterns (MAMPs), which are molecules ormolecular motifs associated with microbes. An exemplary MAMP includes,but is not limited to, lipopolysaccharide (LPS) or an endotoxin.

The microbe-binding magnetic particles comprise on their surfacemicrobe-binding molecules. A microbe-binding molecule can comprise atleast one (e.g., one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty or more) microbe surface-binding domain(“microbe binding domain”). The term “microbe surface-binding domain” asused herein refers to any molecules or a fragment thereof that canspecifically bind to the surface of a microbe, e.g., any componentpresent on a surface of a microbe, or a MAMP.

Any molecule or material that can bind to a microbe or MAMP can beemployed as the microbe-binding molecule. Materials or substances whichcan serve as microbe-binding molecules include, for example, peptides,polypeptides, proteins, peptidomimetics, antibodies, antibody fragments(e.g., antigen binding fragments of antibodies), carbohydrate-bindingprotein, e.g., a lectin, glycoproteins, glycoprotein-binding molecules,amino acids, carbohydrates (including mono-, di-, tri- andpoly-saccharides), lipids, steroids, hormones, lipid-binding molecules,cofactors, nucleosides, nucleotides, nucleic acids (e.g., DNA or RNA,analogues and derivatives of nucleic acids, or aptamers), peptidoglycan,lipopolysaccharide, small molecules, and any combinations thereof. Themicrobe-binding molecule can be covalently (e.g., cross-linked) ornon-covalently linked to the magnetic particles.

In some embodiments, the microbe-binding molecule can comprise anopsonin or a fragment thereof. The term “opsonin” as used herein refersto naturally-occurring and synthetic molecules which are capable ofbinding to or attaching to the surface of a microbe or a pathogen, ofacting as binding enhancers for a process of phagocytosis. Examples ofopsonins which can be used in the engineered molecules described hereininclude, but are not limited to, vitronectin, fibronectin, complementcomponents such as C1q (including any of its component polypeptidechains A, B and C), complement fragments such as C3d, C3b and C4b,mannose-binding protein, conglutinin, surfactant proteins A and D,C-reactive protein (CRP), alpha2-macroglobulin, and immunoglobulins, forexample, the Fc portion of an immunoglobulin.

In some embodiments, the microbe-binding molecule comprises acarbohydrate recognition domain or a carbohydrate recognition portionthereof. As used herein, the term “carbohydrate recognition domain”refers to a region, at least a portion of which, can bind tocarbohydrates on a surface of a microbe (e.g., a pathogen) or a MAMP.

In some embodiments, the microbe-binding molecule comprises at least amicrobial-binding portion of C-type lectins, collectins, ficolins,receptor-based lectins, lectins from the shrimp Marsupenaeus japonicas,non-C-type lectins, lipopolysaccharide (LPS)-binding proteins,endotoxin-binding proteins, peptidoglycan-binding proteins, or anycombinations thereof. In some embodiments, the microbe-binding moleculesis selected from the group consisting of mannose-binding lectin (MBL),surfactant protein A, surfactant protein D, collectin 11, L-ficolin,ficolin A, DC-SIGN, DC-SIGNR, SIGNR1, macrophage mannose receptor 1,dectin-1, dectin-2, lectin A, lectin B, lectin C, wheat germ agglutinin,CD14, MD2, lipopolysaccharide-binding protein (LBP), limulus anti-LPSfactor (LAL-F), mammalian peptidoglycan recognition protein-1 (PGRP-1),PGRP-2, PGRP-3, PGRP-4, C-reactive protein (CRP), or any combinationsthereof.

In some embodiments, the microbe-binding molecule comprises a lectin ora carbohydrate recognition or binding fragment or portion thereof. Theterm “lectin” as used herein refers to any molecules including proteins,natural or genetically modified, that interact specifically withsaccharides (i.e., carbohydrates). The term “lectin” as used herein canalso refer to lectins derived from any species, including, but notlimited to, plants, animals, insects and microorganisms, having adesired carbohydrate binding specificity. Examples of plant lectinsinclude, but are not limited to, the Leguminosae lectin family, such asConA, soybean agglutinin, peanut lectin, lentil lectin, and Galanthusnivalis agglutinin (GNA) from the Galanthus (snowdrop) plant. Otherexamples of plant lectins are the Gramineae and Solanaceae families oflectins. Examples of animal lectins include, but are not limited to, anyknown lectin of the major groups S-type lectins, C-type lectins, P-typelectins, and I-type lectins, and galectins. In some embodiments, thecarbohydrate recognition domain can be derived from a C-type lectin, ora fragment thereof. C-type lectin can include any carbohydrate-bindingprotein that requires calcium for binding. In some embodiments, theC-type lectin can include, but are not limited to, collectin, DC-SIGN,and fragments thereof. Without wishing to be bound by theory, DC-SIGNcan generally bind various microbes by recognizinghigh-mannose-containing glycoproteins on their envelopes and/or functionas a receptor for several viruses such as HIV and Hepatitis C.

In some embodiments, the microbe-binding molecules can comprise amicrobe-binding portion of the C-type lectins, including, e.g., but notlimited to, soluble factors such as Collectins (e.g., MBL, surfactantprotein A, surfactant protein D and Collectin 11), ficolins (e.g.L-Ficolin, Ficolin A), receptor based lectins (e.g., DC-SIGN, DC-SIGNR,SIGNR1, Macrophage Mannose Receptor 1, Dectin-1 and Dectin-2), lectinsfrom the shrimp Marsupenaeus japonicus (e.g., Lectin A, Lectin B andLectin C), or any combinations thereof.

In some embodiments, the microbe-binding molecules can comprise at leasta portion of non-C-type lectins (e.g., but not limited to, Wheat GermAgglutinin).

In some embodiments, the microbe-binding molecules can comprise at leasta portion of lipopolysaccharide (LPS)-binding proteins and/or endotoxinbinding proteins (e.g., but not limited to, CD14, MD2,lipopolysaccharide binding proteins (LBP), limulus anti-LPS factor(LAL-F), or any combinations thereof).

In some embodiments, the microbe-binding molecules can comprise at leasta portion of peptidoglycan binding proteins (e.g., but not limited to,mammalian peptidoglycan recognition protein-1 (PGRP-1), PGRP-2, PGRP-3,PGRP-4, or any combinations thereof.

In some embodiments, the microbe-binding molecules comprise the fullamino acid sequence of a carbohydrate-binding protein, e.g., a lectinmolecule. In some embodiments, the microbe-binding molecules aregenetically engineered to remove a domain that activates the complementsystem and/or binds

In some embodiments, the microbe-binding molecule comprises amannose-binding lectin (MBL) or a carbohydrate binding fragment orportion thereof. Mannose-binding lectin, also called mannose bindingprotein (MBP), is a calcium-dependent serum protein that can play a rolein the innate immune response by binding to carbohydrates on the surfaceof a wide range of microbes or pathogens (viruses, bacteria, fungi,protozoa) where it can activate the complement system. MBL can alsoserve as a direct opsonin and mediate binding and uptake of microbes orpathogens by tagging the surface of a microbe or pathogen to facilitaterecognition and ingestion by phagocytes. Full length MBL and anengineered form of MBL (FcMBL and AKT-FcMBL) are described in theInternational Application Publication Nos. WO/2011/090954 andWO/2013/012924, contents of both of which are incorporated herein byreference.

Without wishing to be bound by a theory, microbe binding moleculescomprising lectins or modified versions thereof can act asbroad-spectrum microbe binding molecules (e.g., pathogen bindingmolecules). Accordingly, the method utilizing lectins (e.g., MBL andgenetically engineered version of MBL (FcMBL and Akt-FcMBL)) asbroad-spectrum microbe binding molecules (e.g., pathogen bindingmolecules) to capture microbes or MAMPs, can be carried out withoutidentifying the microbe (e.g., pathogen).

In some embodiments, at least two (e.g. two, three, four, five, six,seven or more) microbe binding molecules can be linked together to forma multimeric microbe-binding molecule.

Any art-recognized recombinant carbohydrate-binding proteins orcarbohydrate recognition domains can also be used in the microbe-bindingmolecules. For example, recombinant mannose-binding lectins, e.g., butnot limited to, the ones disclosed in the U.S. Pat. Nos. 5,270,199;6,846,649; and U.S. Patent Application No. US 2004/0,229,212, content ofboth of which is incorporated herein by reference, can be used inconstructing a microbe-binding molecule.

In some embodiments, microbe-binding molecules and microbe-bindingmagnetic particles described in the International ApplicationPublication Nos. WO/2011/090954 and WO/2013/012924, contents of both ofwhich are incorporated herein by reference, can be used in the methoddescribed herein. For example, in some embodiments, the microbe-bindingmolecules can be selected from the group consisting of MBL (mannosebinding lectin), FcMBL (IgG Fc fused to mannose binding lectin),AKT-FcMBL (IgG Fc-fused to mannose binding lectin with the N-terminalamino acid tripeptide of sequence AKT (alanine, lysine, threonine)), andany combination thereof, as described in the International ApplicationPublication Nos. WO/2011/090954 and WO/2013/012924, contents of both ofwhich are incorporated herein by reference.

In some embodiments, the microbe-binding molecules each comprise anamino acid sequence selected from SEQ ID NO. 1-SEQ ID NO. 8, wherein theamino acid sequences are shown as follows:

MBL full length (SEQ ID NO. 1):MSLFPSLPLL LLSMVAASYS ETVTCEDAQK TCPAVIACSSPGINGFPGKD GRDGTKGEKG EPGQGLRGLQ GPPGKLGPPGNPGPSGSPGP KGQKGDPGKS PDGDSSLAAS ERKALQTEMARIKKWLTFSL GKQVGNKFFL TNGEIMTFEK VKALCVKFQASVATPRNAAE NGAIQNLIKE EAFLGITDEK TEGQFVDLTGNRLTYTNWNE GEPNNAGSDE DCVLLLKNGQ WNDVPCSTSH LAVCEFPIMBL without the signal sequence (SEQ ID NO. 2):ETVTCEDAQK TCPAVIACSS PGINGFPGKD GRDGTKGEKGEPGQGLRGLQ GPPGKLGPPG NPGPSGSPGP KGQKGDPGKSPDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFLTNGEIMTFEK VKALCVKFQA SVATPRNAAE NGAIQNLIKEEAFLGITDEK TEGQFVDLTG NRLTYTNWNE GEPNNAGSDEDCVLLLKNGQ WNDVPCSTSH LAVCEFPI Truncated MBL (SEQ ID NO. 3):AASERKALQT EMARIKKWLT FSLGKQVGNK FFLTNGEIMTFEKVKALCVK FQASVATPRN AAENGAIQNL IKEEAFLGITDEKTEGQFVD LTGNRLTYTN WNEGEPNNAG SDEDCVLLLK NGQWNDVPCS TSHLAVCEFP ICarbohydrate recognition domain (CRD) of MBL (SEQ ID NO. 4):VGNKFFLTNG EIMTFEKVKA LCVKFQASVA TPRNAAENGAIQNLIKEEAF LGITDEKTEG QFVDLTGNRL TYTNWNEGEPNNAGSDEDCV LLLKNGQWND VPCSTSHLAV CEFPINeck+ Carbohydrate recognition domain of MBL (SEQ ID NO. 5):PDGDSSLAAS ERKALQTEMA RIKKWLTFSL GKQVGNKFFLTNGEIMTFEK VKALCVKFQA SVATPRNAAE NGAIQNLIKEEAFLGITDEK TEGQFVDLTG NRLTYTNWNE GEPNNAGSDEDCVLLLKNGQ WNDVPCSTSH LAVCEFPI FcMBL.81 (SEQ ID NO. 6):EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSPGAPDGDSSLAASERKALQTE MARIKKWLTF SLGKQVGNKFFLTNGEIMTF EKVKALCVKF QASVATPRNA AENGAIQNLIKEEAFLGITD EKTEGQFVDL TGNRLTYTNW NEGEPNNAGSDEDCVLLLKN GQWNDVPCST SHLAVCEFPI Akt-FcMBL (SEQ ID NO. 7):AKTEPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSP GAPDGDSSLAASERKALQTE MARIKKWLTF SLGKQVGNKF FLTNGEIMTFEKVKALCVKF QASVATPRNA AENGAIQNLI KEEAFLGITDEKTEGQFVDL TGNRLTYTNW NEGEPNNAGS DEDCVLLLKN GQWNDVPCST SHLAVCEFPIFcMBL.111 (SEQ ID NO. 8): EPKSSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSP GATSKQVGNKFFLTNGEIMTF EKVKALCVKF QASVATPRNA AENGAIQNLIKEEAFLGITD EKTEGQFVDL TGNRLTYTNW NEGEPNNAGSDEDCVLLLKN GQWNDVPCST SHLAVCEFPI

The microbe-binding molecules can contain sequences from the samespecies or from different species. For example, an interspecies hybridmicrobe-binding molecule can contain a linker, e.g., a peptide linker,from a murine species, and a human sequence from a carbohydraterecognition domain protein, provided that they do not provideunacceptable levels of deleterious effects. The engineeredmicrobe-binding molecules described herein can also include those thatare made entirely from murine-derived sequences or fully human.

In some embodiments, the microbe-binding molecule can be linked to theC-terminal of a linker, e.g., a peptide linker. An exemplary peptidelinker includes, but is not limited to, an Fc portion of animmunoglobulin.

General methods of preparing such microbe-binding molecules are wellknown in the art (Ashkenazi, A. and S. M. Chamow (1997), “Immunoadhesinsas research tools and therapeutic agents,” Curr. Opin. Immunol. 9(2):195-200, Chamow, S. M. and A. Ashkenazi (1996). “Immunoadhesins:principles and applications,” Trends Biotechnol. 14(2):52-60). In oneexample, an engineered microbe-binding molecule can be made by cloninginto an expression vector such as Fc-X vector as discussed in Lo et al.(1998) 11:495 and PCT application no. PCT/US2011/021603, filed Jan. 19,2011, content of both of which is incorporated herein by reference.

Target Molecules or Target Species to be Captured or Removed by theMethods, Kits, Devices, and Solid Substrates Described Herein

The methods, devices, kits and solid substrates described herein can beused to capture, separate, or isolate one or more target molecules orspecies from a test sample. As used interchangeably herein, the term“target species” or “target molecules” refers to any molecule, cell orparticulate that is to be separated or isolated from a fluid sample.Representative examples of target cellular species include, but are notlimited to, mammalian cells, viruses, bacteria, fungi, yeast, protozoan,microbes, and parasites. Representative examples of target moleculesinclude, but are not limited to, hormones, growth factors, cytokines(e.g., inflammatory cytokines), proteins, peptides, prions, lectins,oligonucleotides, carbohydrates, lipids, exosomes, contaminatingmolecules and particles, molecular and chemical toxins, and MAMPs. Thetarget species can also include contaminants found in non-biologicalfluids, such as pathogens or lead in water or in petroleum products.Parasites can include organisms within the phyla Protozoa,Platyhelminthes, Aschelminithes, Acanthocephala, and Arthropoda.

In some embodiments, the target species can include a biological cellselected from the group consisting of living or dead cells (prokaryoticand eukaryotic, including mammalian), viruses, bacteria, fungi, yeast,protozoan, microbes, and parasites. The biological cell can be a normalcell or a diseased cell, e.g., a cancer cell. Mammalian cells include,without limitation; primate, human and a cell from any animal ofinterest, including without limitation; mouse, hamster, rabbit, dog,cat, domestic animals, such as equine, bovine, murine, ovine, canine,and feline. In some embodiments, the cells can be derived from a humansubject. In other embodiments, the cells are derived from a domesticatedanimal, e.g., a dog or a cat. Exemplary mammalian cells include, but arenot limited to, stem cells, cancer cells, progenitor cells, immunecells, blood cells, fetal cells, and any combinations thereof. The cellscan be derived from a wide variety of tissue types without limitationsuch as; hematopoietic, neural, mesenchymal, cutaneous, mucosal,stromal, muscle, spleen, reticuloendothelial, epithelial, endothelial,hepatic, kidney, gastrointestinal, pulmonary, cardiovascular, T-cells,and fetus. Stem cells, embryonic stem (ES) cells, ES-derived cells,induced pluripotent stem cells, and stem cell progenitors are alsoincluded, including without limitation, hematopoietic, neural, stromal,muscle, cardiovascular, hepatic, pulmonary, and gastrointestinal stemcells. Yeast cells may also be used as cells in some embodimentsdescribed herein. In some embodiments, the cells can be ex vivo orcultured cells, e.g. in vitro. For example, for ex vivo cells, cells canbe obtained from a subject, where the subject is healthy and/or affectedwith a disease. While cells can be obtained from a fluid sample, e.g., ablood sample, cells can also be obtained, as a non-limiting example, bybiopsy or other surgical means know to those skilled in the art.

In some embodiments, the target molecules to be captured or removed froma fluid by the method described herein comprise microbes. As usedherein, the term “microbes” generally refer to microorganisms, includingbacteria, fungi, protozoan, archaea, protists, e.g., algae, and acombination thereof. The term “microbes” also includes pathogenicmicrobes, e.g., bacteria causing diseases such as plague, tuberculosisand anthrax; protozoa causing diseases such as malaria, sleepingsickness and toxoplasmosis; fungi causing diseases such as ringworm,candidiasis or histoplasmosis; and bacteria causing diseases such assepsis. The term “microbe” or “microbes” can also encompassnon-pathogenic microbes, e.g., some microbes used in industrialapplications.

In some embodiments, the term “microbe” or “microbes” encompasses intactmicrobes and fragments of microbes, e.g., cell components of microbes,and MAMPs, e.g., lipopolysaccharide (LPS), and/or endotoxin.

Exemplary fungi and yeast include, but are not limited to, Cryptococcusneoformans, Candida albicans, Candida tropicalis, Candida stellatoidea,Candida glabrata, Candida krusei, Candida parapsilosis, Candidaguilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorulamucilaginosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillusclavatus, Cryptococcus neoformans, Cryptococcus laurentii, Cryptococcusalbidus, Cryptococcus gattii, Histoplasma capsulatum, Pneumocystisjirovecii (or Pneumocystis carinii), Stachybotrys chartarum, and anycombination thereof.

Exemplary bacteria include, but are not limited to: anthrax,campylobacter, cholera, diphtheria, enterotoxigenic E. coli, giardia,gonococcus, Helicobacter pylori, Hemophilus influenza B, Hemophilusinfluenza non-typable, meningococcus, pertussis, pneumococcus,salmonella, shigella, Streptococcus B, group A Streptococcus, tetanus,Vibrio cholerae, yersinia, Staphylococcus, Pseudomonas species,Clostridia species, Myocobacterium tuberculosis, Mycobacterium leprae,Listeria monocytogenes, Salmonella typhi, Shigella dysenteriae, Yersiniapestis, Brucella species, Legionella pneumophila, Rickettsiae,Chlamydia, Clostridium perfringens, Clostridium botulinum,Staphylococcus aureus, Treponema pallidum, Haemophilus influenzae,Treponema pallidum, Klebsiella pneumoniae, Pseudomonas aeruginosa,Cryptosporidium parvum, Streptococcus pneumoniae, Bordetella pertussis,Neisseria meningitides, and any combination thereof.

Exemplary parasites include, but are not limited to: Entamoebahistolytica; Plasmodium species, Leishmania species, Toxoplasmosis,Helminths, and any combination thereof.

Exemplary viruses include, but are not limited to, HIV-1, HIV-2,hepatitis viruses (including hepatitis B and C), Ebola virus, West Nilevirus, and herpes virus such as HSV-2, adenovirus, dengue serotypes 1 to4, ebola, enterovirus, herpes simplex virus 1 or 2, influenza, Japaneseequine encephalitis, Norwalk, papilloma virus, parvovirus B19, rubella,rubeola, vaccinia, varicella, Cytomegalovirus, Epstein-Barr virus, Humanherpes virus 6, Human herpes virus 7, Human herpes virus 8, Variolavirus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus,Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, poliovirus,Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measlesvirus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus,Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Rabies virus,Rous sarcoma virus, Yellow fever virus, Ebola virus, Marburg virus,Lassa fever virus, Eastern Equine Encephalitis virus, JapaneseEncephalitis virus, St. Louis Encephalitis virus, Murray Valley fevervirus, West Nile virus, Rift Valley fever virus, Rotavirus A, RotavirusB, Rotavirus C, Sindbis virus, Human T-cell Leukemia virus type-1,Hantavirus, Rubella virus, Simian Immunodeficiency viruses, and anycombination thereof.

In some embodiments, the method described herein can be used to capture,separate, or remove bioterror agents (e.g., B. Anthracis, and smallpox)from a fluid.

Exemplary contaminants found in non-biological fluids can include, butare not limited to microorganisms (e.g., Cryptosporidium, Giardialamblia, bacteria, Legionella, Coliforms, viruses, fungi), bromates,chlorites, haloactic acids, trihalomethanes, chloramines, chlorine,chlorine dioxide, antimony, arsenic, mercury (inorganic), nitrates,nitrites, selenium, thallium, Acrylamide, Alachlor, Atrazine, Benzene,Benzo(a)pyrene (PAHs), Carbofuran, Carbon, etrachloride, Chlordane,Chlorobenzene, 2,4-D, Dalapon, 1,2-Dibromo-3-chloropropane (DBCP),o-Dichlorobenzene, p-Dichlorobenzene, 1,2-Dichloroethane,1,1-Dichloroethylene, cis-1,2-Dichloroethylene,trans-1,2-Dichloroethylene, Dichloromethane, 1,2-Dichloropropane,Di(2-ethylhexyl) adipate, Di(2-ethylhexyl) phthalate, Dinoseb, Dioxin(2,3,7,8-TCDD), Diquat, Endothall, Endrin, Epichlorohydrin,Ethylbenzene, Ethylene dibromide, Glyphosate, Heptachlor, Heptachlorepoxide, Hexachlorobenzene, Hexachlorocyclopentadiene, Lead, Lindane,Methoxychlor, Oxamyl (Vydate), Polychlorinated, biphenyls (PCBs),Pentachlorophenol, Picloram, Simazine, Styrene, Tetrachloroethylene,Toluene, Toxaphene, 2,4,5-TP (Silvex), 1,2,4-Trichlorobenzene,1,1,1-Trichloroethane, 1,1,2-Trichloroethane, Trichloroethylene, Vinylchloride, and Xylenes.

In some embodiments, the target species refers to a rare cell or acellular component thereof. In some embodiments, the target species canrefer to a rare cell or a cellular component thereof derived from amammalian subject, including, without limitation, primate, human or anyanimal of interest such as mouse, hamster, rabbit, dog, cat, domesticanimals, such as equine, bovine, murine, ovine, canine, and feline. Insome embodiments, the rare cells can be derived from a human subject. Inother embodiments, the rare cells can be derived from a domesticatedanimal or a pet such as a cat or a dog. As used herein, the term “rarecells” is defined, in some embodiments, as cells that are not normallypresent in a fluid sample, e.g., a biological fluid sample, but can bepresent as an indicator of an abnormal condition, such as infectiousdisease, chronic disease, injury, proliferative diseases, or pregnancy.In some embodiments, the term “rare cells” as used herein refers tocells that can be normally present in biological specimens, but arepresent with a frequency several orders of magnitude (e.g., at leastabout 100-fold, at least about 1000-fold, at least about 10000-fold)less than other cells typically present in a normal biological specimen.In some embodiments, rare cells are found infrequently in circulatingblood, e.g., less than 100 cells (including less than 10 cells, lessthan 1 cell) per 10⁸ mononuclear cells in about 50 mL of peripheralblood. In some embodiments, a rare cell can be a normal cell or adiseased cell. Examples of rare cells include, but are not limited to,circulating tumor cells, progenitor cells, e.g., collected for bonemarrow transplantation, blood vessel-forming progenitor cells, stemcells, circulating fetal cells, e.g., in maternal peripheral blood forprenatal diagnosis, virally-infected cells, cell subsets collected andmanipulated for cell and gene therapy, and cell subpopulations purifiedfor subsequent gene expression or proteomic analysis, other cellsrelated to disease progression, and any combinations thereof.

As used herein, the term “a cellular component” in reference tocirculating tumor cells, stem cells, fetal cells and/or microbes isintended to include any component of a cell that can be at leastpartially isolated from the cell, e.g., upon lysis of the cell. Cellularcomponents can include, but are not limited to, organelles, such asnuclei, perinuclear compartments, nuclear membranes, mitochondria,chloroplasts, or cell membranes; polymers or molecular complexes, suchas lipids, polysaccharides, proteins (membrane, trans-membrane, orcytosolic); nucleic acids, viral particles, or ribosomes; or othermolecules, such as hormones, ions, and cofactors.

As used herein, the term “cytokine” can refer to any smallcell-signaling protein molecule that is secreted by a cell of any type.Cytokines can include proteins, peptides, and/or glycoproteins. Based ontheir function, cell of secretion, and/or target of action, cytokinescan be generally classified as lymphokines, interleukins, andchemokines. The term “lymphokines” as used herein generally refers to asubset of cytokines that are produced by a type of immune cell known asa lymphocyte. The term “interleukins” as used herein generally refers tocytokines secreted and/or synthesized by leukocytes and helper CD4+Tlymphocytes, and/or through monocytes, macrophages, and/or endothelialcells. In some embodiments, interleukins can be human interleukinsincluding IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,IL-31, IL-32, IL-33, IL-34, and IL-35. The term “chemokine” as usedherein generally refers to a specific class of cytokines that mediateschemoattraction (chemotaxis) between cells. Examples of chemokinesinclude, but are not limited to, CCL family, CXCL family, CX3CL familyand XCL family.

The term “inflammatory cytokine” as used herein generally includes,without limitation, a cytokine that stimulates an inflammatory response.Examples of inflammatory cytokines include, without limitation, IFN-γ,IL-1, and TNF-α.

As used herein, the term “hormone” can refer to polypeptide hormones,which are generally secreted by glandular organs with ducts. Includedamong the hormones are, for example, growth hormone such as human growthhormone, N-methionyl human growth hormone, and bovine growth hormone;parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; estradiol;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, or testolactone; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); prolactin, placental lactogen, mousegonadotropin-associated peptide, gonadotropin-releasing hormone;inhibin; activin; mullerian-inhibiting substance; and thrombopoietin. Asused herein, the term hormone includes proteins from natural sources orfrom recombinant cell culture and biologically active equivalents of thenative-sequence hormone, including synthetically produced small-moleculeentities and pharmaceutically acceptable derivatives and salts thereof.

The term “growth factor” as used herein can refer to proteins thatgenerally promote growth, and include, for example, hepatic growthfactor; fibroblast growth factor; vascular endothelial growth factor;nerve growth factors such as NGF-β; platelet-derived growth factor;transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-likegrowth factor-I and —II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-α, -β, and -γ; and colony stimulatingfactors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF). Asused herein, the term growth factor includes proteins from naturalsources or from recombinant cell culture and biologically activeequivalents of the native-sequence growth factor, includingsynthetically produced small-molecule entities and pharmaceuticallyacceptable derivatives and salts thereof.

As used herein, the term “molecular toxin” refers to a compound producedby an organism which causes or initiates the development of a noxious,poisonous or deleterious effect in a host presented with the toxin. Suchdeleterious conditions may include fever, nausea, diarrhea, weight loss,neurologic disorders, renal disorders, hemorrhage, and the like. Toxinsinclude, but are not limited to, bacterial toxins, such as choleratoxin, heat-liable and heat-stable toxins of E. coli, toxins A and B ofClostridium difficile, aerolysins, and hemolysins; toxins produced byprotozoa, such as Giardia; toxins produced by fungi. Molecular toxinscan also include exotoxins, i.e., toxins secreted by an organism as anextracellular product, and enterotoxins, i.e., toxins present in the gutof an organism.

Example Applications and Fluid Suitable for the Methods, Kits, Devices,and Compositions Described Herein

The methods, kits, devices, and solid substrates described herein areversatile and can be adapted to capture, separate, or remove any targetmolecules from any fluid, depending on the desired application ofinterest. Thus, fluids of any sources can be brought into contact with amagnetic capture surface as described herein or introduced into amagnetic separation chamber as described herein. For example, the fluidcan be a biological fluid obtained or derived from a subject, a fluid orspecimen obtained from an environmental source, a fluid from a cellculture, a microbe colony, or any combinations thereof.

In one embodiment, the methods, kits, devices, and solid substratesdescribed herein can be used to capture, separate, or remove cells(including, e.g., rare cells such as circulating tumor cells ormicrobes) from a fluid of a subject and/or analyze the captured cellsfor therapeutic and/or diagnostic applications. Accordingly, in someembodiments, the fluid is a biological fluid selected from blood,plasma, cord blood, serum, lactation products, amniotic fluids, sputum,saliva, urine, semen, cerebrospinal fluid, bronchial aspirate, bronchiallavage aspirate fluid, perspiration, mucus, liquefied stool sample,synovial fluid, peritoneal fluid, pleural fluid, pericardial fluid,lymphatic fluid, tears, tracheal aspirate, a homogenate of a tissuespecimen, or any mixtures thereof.

In some embodiments, the methods, kits, devices, and solid substratesdescribed herein can be used to remove microbes and/or MAMPs from afluid (e.g., blood or cord blood) of a subject or to purify a fluid(e.g., blood or cord blood) of a subject prior to preservation (e.g.,cryopreservation) and/or transplantation. For example, one of thechallenges in cryopreservation of cord blood is that 5-7% of cord bloodsamples are contaminated by pathogens (mostly E. coli), whichpotentially cause adverse effects on stem cells preserved in cord blood.Thus, treating cord blood to remove pathogenic contaminants using themethod described herein is a beneficial step prior to a cryopreservationprocess.

In some embodiments, the methods, kits, devices, and solid substratesdescribed herein can be used for continuous separation of cells orbiomolecules from blood in an extracorporeal setup. For example, themethods, kits, devices, and solid substrates described herein can beused in blood dialysis of blood to remove microbes and/or toxins.

In some embodiments, the methods, kits, devices, and solid substratesdescribed herein can be used to purify or clean a non-body ornon-biological fluid, e.g., to remove target molecules from the non-bodyfluid. For example, the methods, kits, devices, and solid substratesdescribed herein can be adapted to purify food products and water, e.g.,to remove microbes, toxins, chemicals, and combinations thereof, in acontinuous or batch process. Another example is to capture microbesand/or toxins from food, medium from microbial cultures (e.g.,pharmaceutical manufacturing, beer brewing, etc.), water, or any otherfluid. Accordingly, in some embodiments, the fluid is a fluid orspecimen obtained from an environmental source selected from a fluid orspecimen obtained or derived from food products, food produce, poultry,meat, fish, beverages, dairy product, water (including wastewater),ponds, rivers, reservoirs, swimming pools, soils, food processing and/orpackaging plants, agricultural places, hydrocultures (includinghydroponic food farms), pharmaceutical manufacturing plants, animalcolony facilities, beer brewing, or any combinations thereof.

As used herein, the term “non-biological fluid” refers to any fluid thatis not a biological fluid as the term is defined herein. Exemplarynon-biological fluids include, but are not limited to, water, saltwater, brine, buffered solutions, saline solutions, sugar solutions,carbohydrate solutions, lipid solutions, nucleic acid solutions,hydrocarbons (e.g. liquid hydrocarbons), acids, gasolines, petroleum,liquefied samples (e.g., liquefied samples), and mixtures thereof.

In some embodiments, the fluid can include a fluid (e.g., culturemedium) from a biological culture. Examples of a fluid (e.g., culturemedium) obtained from a biological culture includes the one obtainedfrom culturing or fermentation, for example, of single- or multi-cellorganisms, including prokaryotes (e.g., bacteria) and eukaryotes (e.g.,animal cells, plant cells, yeasts, fungi), and including fractionsthereof. In some embodiments, the fluid can include a fluid from a bloodculture. In some embodiments, the culture medium can be obtained fromany source, e.g., without limitations, research laboratories,pharmaceutical manufacturing plants, hydrocultures (e.g., hydroponicfood farms), diagnostic testing facilities, clinical settings, and anycombinations thereof.

The fluid, including any fluid or specimen (processed or unprocessed)can be liquid, supercritical fluid, solutions, suspensions, gases, gels,slurries, and combinations thereof. The fluid can be aqueous ornon-aqueous. In some embodiments, the fluid can be an aqueous fluid. Asused herein, the term “aqueous fluid” refers to any flowablewater-containing material that is suspected of comprising a pathogen.

In some embodiments, the fluid can include a media or reagent solutionused in a laboratory or clinical setting, such as for biomedical andmolecular biology applications. As used herein, the term “media” refersto a medium for maintaining a tissue, an organism, or a cell population,or refers to a medium for culturing a tissue, an organism, or a cellpopulation, which contains nutrients that maintain viability of thetissue, organism, or cell population, and support proliferation andgrowth.

As used herein, the term “reagent” refers to any solution used in alaboratory or clinical setting for biomedical and molecular biologyapplications. Reagents include, but are not limited to, salinesolutions, PBS solutions, buffered solutions, such as phosphate buffers,EDTA, Tris solutions, and any combinations thereof. Reagent solutionscan be used to create other reagent solutions. For example, Trissolutions and EDTA solutions are combined in specific ratios to create“TE” reagents for use in molecular biology applications.

In some embodiments, the methods, kits, devices, and solid substratesdescribed herein are used to remove sepsis related target componentsfrom the blood of a subject in need thereof. As used herein, sepsisrelated target components refer to any molecule or bioparticle that cancontribute to development of sepsis in a subject.

As used herein, “sepsis” refers to a body or subject's response to asystemic microbial infection. Sepsis is the leading cause of death ofimmunocompromised patients, and is responsible for over 200,000 deathsper year in the United States. The onset of sepsis occurs when rapidlygrowing infectious agents saturate the blood and overcome a subject'simmunological clearance mechanisms. Most existing therapies areineffective, and subjects can die because of clot formation,hypoperfusion, shock, and multiple organ failure.

In some embodiments, the methods, kits, devices, and solid substratesdescribed herein are used to in combination with conventional therapiesfor treating a subject in need thereof. For example, the methods, kits,devices, and solid substrates described herein are used in conjunctionwith conventional therapies for sepsis treatment, such as fungicides. Inanother example, the methods, kits, devices, and solid substratesdescribed herein are used for treating a subject having a cancer. Insome embodiments, the methods, kits, devices, and solid substratesdescribed herein can be used to remove cancer cells from a biologicalfluid obtained from the subject, and to provide an additional treatmentincluding, but not limited to, chemotherapy, radiation therapy,steroids, bone marrow transplants, stem cell transplants, growth factoradministration, ATRA (all-trans-retinoic acid) administration, histaminedihydrochloride (Ceplene) administration, interleukin-2 (Proleukin)administration, gemtuzumab ozogamicin (Mylotarg) administration,clofarabine administration, farnesyl transferase inhibitoradministration, decitabine administration, inhibitor of MDR1(multidrug-resistance protein) administration, arsenic trioxideadministration, rituximab administration, cytarabine (ara-C)administration, anthracycline administration (such as daunorubicin oridarubicin), imatinib administration, dasatanib administration,nilotinib administration, purine analogue (such as fludarabine)administration, alemtuzumab (anti-CD52) administration, (fludarabinewith cyclophosphamide), fludarabine administration, cyclophosphamideadministration, doxorubicin administration, vincristine administration,prednisolone administration, lenalidomide administration, flavopiridoladministration, or any combination therein. In some embodiments, themethods, kits, devices, and solid substrates described herein are usedfor treating a subject in need thereof without providing any othertherapy to the subject. For example, the methods, kits, devices, andsolid substrates described herein are used for sepsis treatment,pathogen and/or toxin clearance from biological fluids, of a subject inneed thereof.

In some embodiments, the methods, kits, devices, and solid substratesdescribed herein are used to purify or enrich a target component from asource fluid. For example, the methods, kits, devices, and solidsubstrates described herein can be used to purify products of chemicalreactions or molecules being produced in a cell culture.

Solid Substrates

A solid substrate comprising a surface having magnetic field gradientconcentrating particles distributed thereon and substantially alignedalong magnetic flux lines of a magnetic field is also described herein.The solid substrate further comprises a target-binding magnetic particleand a target.

In one embodiment, the target is bound to the target-binding magneticparticle.

In some embodiments, the solid substrate is selected from the groupconsisting of a channel, a microfluidic channel, a sample well, amicrotiter plate, a magnetic comb, a slide (e.g., a glass slide), aflask (e.g., a tissue culture flask), a tube, a nanotube, a fiber, afilter, a membrane, a scaffold, an extracorporeal device, a mixer, amicrofluidic device, a hollow fiber, or any combinations thereof.

In some embodiments, the solid substrate can further comprise astructure or device that produces a magnetic field. Thus, the magneticfield gradient concentrating particles can be substantially alignedalong magnetic flux lines of the magnetic field produced by thestructure or device.

In some embodiments, the solid substrate can include a magnet embeddedtherein. For example, a magnetic multi-well tip comb contains a magnetin each tip. See, e.g., FIG. 6A.

In some embodiments, a magnet is placed in contact with or in proximityto at least one surface of the solid substrate. Accordingly, a systemcomprising (a) a magnet; and (b) the solid substrate described herein(comprising the magnetic field gradient concentrating particles, thetarget-binding magnetic particles and the target) is also describedherein.

Kits

Another aspect described herein relates to a kit comprising (i) a devicecomprising a magnetic separation chamber or a magnetic capture surface;(ii) one or more containers containing magnetic field gradientconcentrating particles; and (iii) one or more containers containingtarget-binding magnetic particles.

In some embodiments, the device can further comprise a structure ormodule that produces a magnetic field. In some embodiments, thestructure or module that produces a magnetic field can be detachablefrom at least a portion of the device, e.g., the magnetic separationchamber or a magnetic capture surface.

The device comprising a magnetic separation chamber or a magneticcapture surface can be any fluid container or fluid processing device.For example, the device can be an eppendorf tube, a multi-well plate, aflask (e.g., a tissue culture flask), an extracorporeal device, a mixer,a hollow fiber cartridge, a microfluidic device, or any combinationsthereof.

In some embodiments, the device is a microfluidic device. In oneembodiment, the device can be an organ-on-chip device (e.g., a biospleendevice).

In some embodiments, the device is a multi-well plate (e.g., 96-wellplate).

Generation of Target-Binding Magnetic Particles

The target-binding magnetic particles can be generated by attachingtarget-binding molecules to magnetic particles using any methods knownin the art.

In some embodiments, the target-binding molecules can be attached themagnetic particles via a linker, which is described in detail below. Insome embodiments, the linker can be a peptide linker. An exemplarypeptide linker comprises an Fc portion of an immunoglobulin. In someembodiments, the N-terminus of the linker can comprise an amino acidsequence of AKT (alanine, lysine, threonine).

In some embodiments, the surface of the magnetic particles can befunctionalized to include a coupling molecule for conjugation of thetarget-binding molecules. As used herein, the term “coupling molecule”refers to any molecule or any functional group that is capable ofselectively binding with a microbe surface-binding domain.Representative examples of coupling molecules include, but are notlimited to, antibodies, antigens, lectins, proteins, peptides, nucleicacids (DNA, RNA, PNA and nucleic acids that are mixtures thereof or thatinclude nucleotide derivatives or analogs); receptor molecules, such asthe insulin receptor; ligands for receptors (e.g., insulin for theinsulin receptor); and biological, chemical or other molecules that haveaffinity for another molecule, such as biotin and avidin. The couplingmolecules need not comprise an entire naturally occurring molecule butmay consist of only a portion, fragment or subunit of a naturally ornon-naturally occurring molecule, as for example the Fab fragment of anantibody. The coupling molecule can further comprise a detectable label.The coupling molecule can also encompass various functional groups thatcan couple the substrate to the engineered microbe surface-bindingdomains. Examples of such functional groups include, but are not limitedto, an amino group, a carboxylic acid group, an epoxy group, and a tosylgroup.

The coupling molecule can be conjugated to the surface of magneticparticles covalently or non-covalently using any of the methods known tothose of skill in the art. For example, covalent immobilization can beaccomplished through, for example, silane coupling. See, e.g., Weetall,15 Adv. Mol. Cell Bio. 161 (2008); Weetall, 44 Meths. Enzymol. 134(1976). The covalent interaction between the coupling molecule and thesurface can also be mediated by other art-recognized chemical reactions,such as NHS reaction. The non-covalent interaction between the couplingmolecule and the surface can be formed based on ionic interactions, vander Waals interactions, dipole-dipole interactions, hydrogen bonds,electrostatic interactions, and/or shape recognition interactions.

In certain embodiments, the organic moiety or functional groups can besurface functional groups capable of direct coupling of magneticparticles to target-binding molecules of a user's choice. For example,in some embodiments, the magnetic particles can be functionalized withvarious surface functional groups, e.g., amino groups, carboxylic acidgroups, epoxy groups, tosyl groups, or silica-like groups. Suitablemagnetic particles are commercially available such as from PerSeptiveDiagnostics, Inc. (Cambridge, Mass.); Invitrogen Corp. (Carlsbad,Calif.); Cortex Biochem Inc. (San Leandro, Calif.); and BangsLaboratories (Fishers, Ind.).

In alternative embodiments, the target-binding molecules can beconjugated to the surface of the magnetic particles by a couplingmolecule pair. The term “coupling molecule pair” as used herein refersto the first and second molecules that specifically bind to each other.One member of the binding pair is conjugated to a magnetic particlewhile the second member is conjugated to a target-binding molecule. Asused herein, the phrase “first and second molecules that specificallybind to each other” refers to binding of the first member of thecoupling pair to the second member of the coupling pair with greateraffinity and specificity than to other molecules. Exemplary couplingmolecule pairs include, without limitations, any haptenic or antigeniccompound in combination with a corresponding antibody or binding portionor fragment thereof (e.g., digoxigenin and anti-digoxigenin; mouseimmunoglobulin and goat antimouse immunoglobulin) and nonimmunologicalbinding pairs (e.g., biotin-avidin, biotin-streptavidin), hormone (e.g.,thyroxine and cortisol-hormone binding protein), receptor-receptoragonist, receptor-receptor antagonist (e.g., acetylcholinereceptor-acetylcholine or an analog thereof), IgG-protein A,lectin-carbohydrate, enzyme-enzyme cofactor, enzyme-enzyme inhibitor,and complementary oligonucleotide pairs capable of forming nucleic acidduplexes). The coupling molecule pair can also include a first moleculethat is negatively charged and a second molecule that is positivelycharged.

One non-limiting example of using conjugation with a coupling moleculepair is the biotin-sandwich method. See, e.g., Davis et al., 103 PNAS8155 (2006). The two molecules to be conjugated together arebiotinylated and then conjugated together using tetravalentstreptavidin. In addition, a peptide can be coupled to the 15-amino acidsequence of an acceptor peptide for biotinylation (referred to as AP;Chen et al., 2 Nat. Methods 99 (2005)). The acceptor peptide sequenceallows site-specific biotinylation by the E. Coli enzyme biotin ligase(BirA; Id.). An engineered microbe surface-binding domain can besimilarly biotinylated for conjugation with a solid substrate. Manycommercial kits are also available for biotinylating proteins. Anotherexample for conjugation to a magnetic particle would be to usePLP-mediated bioconjugation. See, e.g., Witus et al., 132 JACS 16812(2010). As described earlier, an AKT sequence on the N terminal of alinker, wherein the C-terminal of the linker is conjugated to atarget-binding molecule, can allow the target-binding molecule to bebiotinylated at a single site and further conjugated to astreptavidin-coated magnetic particle.

Linkers

As used herein, the term “linker” generally refers to a molecular entitythat can directly or indirectly connect two parts of a composition,e.g., at least one target-binding molecule and a magnetic particle.

Linkers can be configures according to a specific need, e.g., based onat least one of the following characteristics. By way of example only,in some embodiments, linkers can be configured to have a sufficientlength and flexibility such that it can allow for a target-bindingmolecule to orient accordingly with respect to a target moleculesurface. In some embodiments, linkers can be configured to allowmultimerization of at least two target-binding molecules (e.g., to froma di-, tri-, tetra-, penta-, or higher multimeric complex) whileretaining biological activity (e.g., microbe-binding activity). In someembodiments, linkers can be configured to facilitate expression andpurification of the target-binding molecule described herein. In someembodiments, linkers can be configured to provide at least onerecognition-site for proteases or nucleases. In addition, linkers shouldbe non-reactive with the functional components of the engineeredmolecule described herein (e.g., minimal hydrophobic or chargedcharacter to react with the functional protein domains such as atarget-binding molecule).

In some embodiments, a linker can be configured to have any length in aform of a peptide, a protein, a nucleic acid (e.g., DNA or RNA), or anycombinations thereof. In some embodiments, the peptide or nucleic acidlinker can vary from about 1 to about 1000 amino acids long, from about10 to about 500 amino acids long, from about 30 to about 300 amino acidslong, or from about 50 to about 150 amino acids long. Longer or shorterlinker sequences can be also used for the target-binding moleculedescribed herein. In one embodiment, the peptide linker has an aminoacid sequence of about 200 to 300 amino acids in length.

In some embodiments, a peptide or nucleic acid linker can be configuredto have a sequence comprising at least one of the amino acids selectedfrom the group consisting of glycine (Gly), serine (Ser), asparagine(Asn), threonine (Thr), methionine (Met) or alanine (Ala), or at leastone of codon sequences encoding the aforementioned amino acids (i.e.,Gly, Ser, Asn, Thr, Met or Ala). Such amino acids and correspondingnucleic acid sequences are generally used to provide flexibility of alinker. However, in some embodiments, other uncharged polar amino acids(e.g., Gln, Cys or Tyr), nonpolar amino acids (e.g., Val, Leu, Ile, Pro,Phe, and Trp), or nucleic acid sequences encoding the amino acidsthereof can also be included in a linker sequence. In alternativeembodiments, polar amino acids or nucleic acid sequence thereof can beadded to modulate the flexibility of a linker. One of skill in the artcan control flexibility of a linker by varying the types and numbers ofresidues in the linker. See, e.g., Perham, 30 Biochem. 8501 (1991);Wriggers et al., 80 Biopolymers 736 (2005).

In alternative embodiments, a linker can be a chemical linker of anylength. In some embodiments, chemical linkers can comprise a direct bondor an atom such as oxygen or sulfur, a unit such as NH, C(O), C(O)NH,SO, SO2, SO2NH, or a chain of atoms, such as substituted orunsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl,substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstitutedC6-C12 aryl, substituted or unsubstituted C5-C12 heteroaryl, substitutedor unsubstituted C5-C12 heterocyclyl, substituted or unsubstitutedC3-C12 cycloalkyl, where one or more methylenes can be interrupted orterminated by O, S, S(O), SO2, NH, or C(O). In some embodiments, thechemical linker can be a polymer chain (branched or linear).

In some embodiments where the linker is a peptide, such peptide linkercan comprise at least a portion of an immunoglobulin, e.g., IgA, IgD,IgE, IgG and IgM including their subclasses (e.g., IgG1), or a modifiedthereof. In some embodiments, the peptide linker can comprise a portionof fragment crystallization (Fc) region of an immunoglobulin or amodified thereof. In such embodiments, the portion of the Fc region thatcan be used as a linker can comprise at least one region selected fromthe group consisting of a hinge region, a CH2 region, a CH3 region, andany combinations thereof. By way of example, in some embodiments, a CH2region can be excluded from the portion of the Fc region as a linker. Inone embodiment, Fc linker comprises a hinge region, a CH2 domain and aCH3 domain. Such Fc linker can be used to facilitate expression andpurification of the target-binding molecule described herein. The Nterminal Fc has been shown to improve expression levels, protein foldingand secretion of the fusion partner. In addition, the Fc has astaphylococcal protein A binding site, which can be used for one-steppurification protein A affinity chromatography. See Lo K M et al. (1998)Protein Eng. 11: 495-500. Further, such Fc linker have a molecule weightabove a renal threshold of about 45 kDa, thus reducing the possibilityof target-binding molecule being removed by glomerular filtration.Additionally, the Fc linker can allow dimerization of two target-bindingmolecule to form a dimer, e.g., a dimeric MBL molecule.

In various embodiments, the N-terminus or the C-terminus of the linker,e.g., the portion of the Fc region, can be modified. By way of exampleonly, the N-terminus or the C-terminus of the linker can be extended byat least one additional linker described herein, e.g., to providefurther flexibility, or to attach additional molecules. In someembodiments, the N-terminus of the linker can be linked directly orindirectly (via an additional linker) with a substrate-binding domainadapted for orienting the carbohydrate recognition domain away from thesubstrate. Exemplary Fc linked MBL (FcMBL and Akt-FcMBL) are describedin PCT application no. PCT/US2011/021603, filed Jan. 19, 2011, contentof which is incorporated herein by reference.

In some embodiments, the linkers can be branched. For branched linkers,the linker can link at least one (e.g., one, two, three, four, five,six, seven, eight, nine, ten or more) target-binding molecule(s) to amagnetic particle.

In some embodiments, the C-terminal of the linker can be conjugated to atarget-binding molecule.

Embodiments of Various Aspects Described Herein can be Defined in any ofthe Following Numbered Paragraphs:

-   1. A method of capturing at least one target from a fluid    comprising:    -   introducing a fluid and target-binding magnetic particles to a        magnetic separation chamber in the presence of a magnetic field        gradient (a gradient of a magnetic field),    -   wherein at least a portion of a fluid-contact surface of the        magnetic separation chamber comprises magnetic field gradient        concentrating particles distributed thereon and substantially        aligned along magnetic flux lines of the magnetic field, and    -   wherein the magnetic field gradient concentrating particles act        as local magnetic field gradient concentrators and attracts at        least a portion of the target-binding magnetic particles to the        magnetic field gradient concentrating particles in the presence        of the magnetic field gradient, whereby a target bound on the        target-binding magnetic particles is captured from the fluid.-   2. The method of paragraph 1, wherein the magnetic field gradient    concentrating particles form magnetic micro- or nano-structures on    said at least a portion of the fluid-contact surface of the magnetic    separation chamber.-   3. The method of paragraph 1 or 2, wherein the magnetic field    gradient concentrating particles comprise superparamagnetic    particles, paramagnetic particles, ferrimagnetic particles,    ferromagnetic particles, or combinations thereof.-   4. The method of any of paragraphs 1-3, wherein the magnetic field    gradient concentrating particles are ferromagnetic particles.-   5. The method of paragraph 4, wherein the ferromagnetic particles    are particles of reduced iron, atomized iron, electrolyte iron,    nickel, cobalt, permalloy, alloy comprising at least one or a    combination of two or more aforementioned ferromagnetic materials,    compounds of rare earth metals, minerals (e.g., iodestone), or a    combination of two or more thereof.-   6. The method of any of paragraphs 1-5, wherein the diameter of the    target-binding magnetic particles is no more than 250 nm, no more    than 100 nm, no more than 50 nm, or no more than 5 nm.-   7. The method of any of paragraphs 1-6, wherein the diameter of the    magnetic field gradient concentrating particles ranges from about 50    nm to about 5 mm.-   8. The method of paragraph 7, wherein the diameter of the magnetic    field gradient concentrating particles is about 300 μm.-   9. The method of any of paragraphs 1-8, wherein at least 50% area or    higher of said at least a portion of the fluid-contact surface    comprises the magnetic field gradient concentrating particles    distributed thereon.-   10. The method of any of paragraphs 1-9 wherein efficiency of    magnetically capturing the target-bound targeting-binding magnetic    particles from the fluid is increased by at least about 50%    (including, e.g., at least about 60%, at least about 70%, at least    about 80%, at least about 90%) or more, as compared to the    efficiency in the absence of the magnetic field concentrating    particles.-   11. The method of any of paragraphs 1-10, wherein efficiency of    magnetically capturing the target-bound targeting-binding magnetic    particles from the fluid is increased by at least about 1.1-fold    (including, e.g., at least about 1.5-fold, at least about 2-fold, at    least about 3-fold, at least about 4-fold) or more, as compared to    the efficiency in the absence of the magnetic field concentrating    particles.-   12. The method of any of paragraphs 1-11, wherein the fluid is    flowed through the magnetic separation chamber at a flow rate of    about 1 ml/hr to about 10 L/hr.-   13. The method of any of paragraphs 1-12, wherein the magnetic    separation chamber comprises a channel, a microfluidic channel, a    sample well, a microtiter plate, a slide (e.g., a glass slide), a    flask (e.g., a tissue culture flask), a tube, a nanotube, a fiber, a    filter, a membrane, a scaffold, an extracorporeal device, a mixer, a    hollow fiber, or any combinations thereof.-   14. The method of any of paragraphs 1-13, wherein the fluid is a    biological fluid obtained or derived from a subject, a fluid or    specimen obtained from an environmental source, a fluid from a cell    culture, a microbe colony, or any combinations thereof.-   15. The method of paragraph 14, wherein the fluid is a biological    fluid selected from blood, plasma, cord blood, serum, lactation    products, amniotic fluids, sputum, saliva, urine, semen,    cerebrospinal fluid, bronchial aspirate, bronchial lavage aspirate    fluid, perspiration, mucus, liquefied stool sample, synovial fluid,    peritoneal fluid, pleural fluid, pericardial fluid, lymphatic fluid,    tears, tracheal aspirate, a homogenate of a tissue specimen, or any    mixtures thereof.-   16. The method of paragraph 14, wherein the fluid is a fluid or    specimen obtained from an environmental source selected from a fluid    or specimen obtained or derived from food products, food produce,    poultry, meat, fish, beverages, dairy product, water (including    wastewater), ponds, rivers, reservoirs, swimming pools, soils, food    processing and/or packaging plants, agricultural places,    hydrocultures (including hydroponic food farms), pharmaceutical    manufacturing plants, animal colony facilities, beer brewing, or any    combinations thereof.-   17. The method of any of paragraphs 1-16, wherein the target-binding    magnetic particles are paramagnetic or superparamagnetic particles.-   18. The method of any of paragraphs 1-17, wherein the target-binding    magnetic particles are magnetic particles adapted to bind a target    selected from the group consisting of cells, proteins, nucleic    acids, microbes, small molecules, chemicals, toxins, drugs, and    combinations thereof.-   19. The method of any of paragraphs 1-18, wherein the target-binding    magnetic particles are microbe-binding magnetic particles.-   20. The method of paragraph 19, wherein the microbe-binding magnetic    particles comprise on their surface microbe-binding molecules.-   21. The method of paragraph 20, wherein the microbe-binding molecule    is selected from the group consisting of opsonins, lectins,    antibodies and antigen binding fragments thereof, proteins,    peptides, peptidomimetics, carbohydrate-binding proteins, nucleic    acids, carbohydrates, lipids, steroids, hormones, lipid-binding    molecules, cofactors, nucleosides, nucleotides, nucleic acids,    peptidoglycan, lipopolysaccharide-binding proteins, small molecules,    and any combination thereof.-   22. The method of paragraph 20 or 21, wherein the microbe-binding    molecule comprises at least a microbial-binding portion of C-type    lectins, collectins, ficolins, receptor-based lectins, lectins from    the shrimp Marsupenaeus japonicas, non-C-type lectins,    lipopolysaccharide (LPS)-binding proteins, endotoxin-binding    proteins, peptidoglycan-binding proteins, or any combinations    thereof.-   23. The method of any of paragraphs 20-22, wherein the    microbe-binding molecule is selected from the group consisting of    mannose-binding lectin (MBL), surfactant protein A, surfactant    protein D, collectin 11, L-ficolin, ficolin A, DC-SIGN, DC-SIGNR,    SIGNR1, macrophage mannose receptor 1, dectin-1, dectin-2, lectin A,    lectin B, lectin C, wheat germ agglutinin, CD14, MD2,    lipopolysaccharide-binding protein (LBP), limulus anti-LPS factor    (LAL-F), mammalian peptidoglycan recognition protein-1 (PGRP-1),    PGRP-2, PGRP-3, PGRP-4, C-reactive protein (CRP), or any    combinations thereof.-   24. The method of any of paragraphs 20-23, wherein the    microbe-binding molecules are attached to the microbe-binding    magnetic particles via a linker.-   25. The method of paragraph 24, wherein the N-terminus of the linker    comprises an amino acid sequence of AKT (alanine, lysine,    threonine).-   26. The method of paragraph 24 or 25, wherein the linker is a    peptide linker.-   27. The method of any of paragraphs 24-26, wherein the linker    comprises a Fc portion of an immunoglobulin.-   28. The method of any of paragraphs 20-27, wherein the    microbe-binding molecule is selected from the group consisting of    MBL (mannose binding lectin), FcMBL (IgG Fc fused to mannose binding    lectin), AKT-FcMBL (IgG Fc-fused to mannose binding lectin with the    N-terminal amino acid tripeptide of sequence AKT (alanine, lysine,    threonine)), and any combination thereof.-   29. The method of any of paragraphs 20-28, wherein the    microbe-binding molecule comprises an amino acid sequence selected    from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID    NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and any combination    thereof.-   30. A kit comprising (i) a device comprising a magnetic separation    surface or chamber; (ii) one or more containers containing magnetic    field gradient concentrating particles; and (iii) one or more    containers containing target-binding magnetic particles.-   31. The kit of paragraph 30, wherein the device is a microfluidic    device (e.g., a biospleen device).-   32. The kit of paragraph 30 or 31, wherein the magnetic field    gradient concentrating particles comprise superparamagnetic    particles, paramagnetic particles, ferrimagnetic particles,    ferromagnetic particles, and combinations thereof.-   33. The kit of any of paragraphs 30-32, wherein the magnetic field    gradient concentrating particles are ferromagnetic particles.-   34. The kit of any of paragraphs 30-33, wherein the target-binding    magnetic particles are magnetic particles adapted or funcationalized    to bind a target selected from the group consisting of cells,    proteins, nucleic acids, microbes, small molecules, chemicals,    toxins, drugs, and combinations thereof.-   35. The kit of any of paragraphs 30-34, wherein the target-binding    magnetic particles are microbe-binding magnetic particles.-   36. The kit of any of paragraphs 30-35, wherein the diameter of the    target-binding magnetic particles is no more than 250 nm, no more    than 100 nm, no more than 50 nm, or no more than 5 nm.-   37. The kit of any of paragraphs 30-36, wherein the diameter of the    magnetic field gradient concentrating particles ranges from about 50    nm to about 5 mm.-   38. A solid substrate comprising (i) a surface having magnetic field    gradient concentrating particles distributed thereon and    substantially aligned along magnetic flux lines of a magnetic    field; (ii) target-binding magnetic particles; and (iii) a target.-   39. The solid substrate of paragraph 38, wherein the target is bound    to at least one of the target-binding magnetic particles.-   40. The solid substrate of paragraph 38 or 39, wherein the diameter    of the magnetic field gradient concentrating particles ranges from    about 50 nm to about 5 mm.-   41. The solid substrate of any of paragraphs 38-40, wherein the    diameter of the target-binding magnetic particles is no more than    250 nm, no more than 100 nm, no more than 50 nm, or no more than 5    nm.-   42. The solid substrate of any of paragraphs 38-41, wherein the    magnetic field gradient concentrating particles comprise    superparamagnetic particles, paramagnetic particles, ferrimagnetic    particles, ferromagnetic particles, and combinations thereof.-   43. The solid substrate of any of paragraphs 38-42, wherein the    magnetic field gradient concentrating particles are ferromagnetic    particles.-   44. The solid substrate of any of paragraphs 38-43, wherein the    target is selected from the group consisting of cells, proteins,    nucleic acids, microbes, small molecules, chemicals, toxins, drugs,    and combinations thereof.-   45. The solid substrate of any of paragraphs 38-44, wherein the    solid substrate is selected from the group consisting of a channel,    a microfluidic channel, a sample well, a microtiter plate, a slide    (e.g., a glass slide), a flask (e.g., a tissue culture flask), a    tube, a nanotube, a fiber, a filter, a membrane, a scaffold, an    extracorporeal device, a mixer, a microfluidic device, a hollow    fiber, or any combinations thereof.-   46. The solid substrate of any of paragraphs 38-45, wherein the    target-binding magnetic particles are microbe-binding magnetic    particles, and the target comprises a microbe.-   47. The solid substrate of paragraph 46, wherein the microbe-binding    magnetic particles comprise each on its surface microbe-binding    molecules.-   48. The solid substrate of paragraph 47, wherein the microbe-binding    molecules are selected from the group consisting of opsonins,    lectins, antibodies and antigen binding fragments thereof, proteins,    peptides, peptidomimetics, carbohydrate-binding proteins, nucleic    acids, carbohydrates, lipids, steroids, hormones, lipid-binding    molecules, cofactors, nucleosides, nucleotides, nucleic acids,    peptidoglycan, lipopolysaccharide-binding proteins, small molecules,    and any combination thereof.-   49. The solid substrate of paragraph 47 or 48, wherein the    microbe-binding molecules each comprises at least a    microbial-binding portion of C-type lectins, collectins, ficolins,    receptor-based lectins, lectins from the shrimp Marsupenaeus    japonicas, non-C-type lectins, lipopolysaccharide (LPS)-binding    proteins, endotoxin-binding proteins, peptidoglycan-binding    proteins, or any combinations thereof.-   50. The solid substrate of any of paragraphs 47-49, wherein the    microbe-binding molecules are each selected from the group    consisting of mannose-binding lectin (MBL), surfactant protein A,    surfactant protein D, collectin 11, L-ficolin, ficolin A, DC-SIGN,    DC-SIGNR, SIGNR1, macrophage mannose receptor 1, dectin-1, dectin-2,    lectin A, lectin B, lectin C, wheat germ agglutinin, CD14, MD2,    lipopolysaccharide-binding protein (LBP), limulus anti-LPS factor    (LAL-F), mammalian peptidoglycan recognition protein-1 (PGRP-1),    PGRP-2, PGRP-3, PGRP-4, C-reactive protein (CRP), or any    combinations thereof.-   51. The solid substrate of any of paragraphs 47-50, wherein the    microbe-binding molecules are attached to the microbe-binding    magnetic particles via a linker.-   52. The solid substrate of paragraph 51, wherein the N-terminus of    the linker comprises an amino acid sequence of AKT (alanine, lysine,    threonine).-   53. The solid substrate of paragraph 51 or 52, wherein the linker is    a peptide linker.-   54. The solid substrate of any of paragraphs 51-53, wherein the    linker comprises a Fc portion of an immunoglobulin.-   55. The solid substrate of any of paragraphs 47-54, wherein the    microbe-binding molecules are each selected from the group    consisting of MBL (mannose binding lectin), FcMBL (IgG Fc fused to    mannose binding lectin), AKT-FcMBL (IgG Fc-fused to mannose binding    lectin with the N-terminal amino acid tripeptide of sequence AKT    (alanine, lysine, threonine)), and any combination thereof.-   56. The solid substrate of any of paragraphs 47-55, wherein the    microbe-binding molecule comprises an amino acid sequence selected    from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID    NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and any combination    thereof.-   57. A method of separating magnetic particles from a fluid    comprising:    -   subjecting a magnetic capture surface and magnetic field        gradient concentrating particles to a magnetic field gradient (a        gradient of a magnetic field), wherein the magnetic field        gradient concentrating particles, in the presence of the        magnetic field gradient, distribute on at least a portion of a        magnetic capture surface and substantially align along magnetic        flux lines of the magnetic field; and    -   contacting the magnetic capture surface with a fluid comprising        magnetic particles, wherein the magnetic field gradient        concentrating particles act as local magnetic field gradient        concentrators, thereby attracting at least a portion of the        magnetic particles to the magnetic field gradient concentrating        particles in the presence of the magnetic field gradient and        separating the magnetic particles from the fluid.-   58. The method of paragraph 57, wherein the magnetic field gradient    concentrating particles form magnetic micro- or nano-structures on    said at least a portion of the magnetic capture surface.-   59. The method of paragraph 57 or 58, wherein the magnetic field    gradient concentrating particles comprise superparamagnetic    particles, paramagnetic particles, ferrimagnetic particles,    ferromagnetic particles, or combinations thereof.-   60. The method of any of paragraphs 57-59, wherein the magnetic    field gradient concentrating particles are ferromagnetic particles.-   61. The method of paragraph 60, wherein the ferromagnetic particles    are particles of reduced iron, atomized iron, electrolyte iron,    nickel, cobalt, permalloy, alloy comprising at least one or a    combination of two or more aforementioned ferromagnetic materials,    compounds of rare earth metals, minerals (e.g., iodestone), or a    combination of two or more thereof.-   62. The method of any of paragraphs 57-61, wherein a dimension    (e.g., diameter) of the magnetic particles is no more than 250 nm,    no more than 100 nm, no more than 50 nm, or no more than 5 nm.-   63. The method of any of paragraphs 57-62, wherein a dimension    (e.g., diameter) of the magnetic field gradient concentrating    particles ranges from about 50 nm to about 5 mm.-   64. The method of paragraph 63, wherein the dimension (e.g.,    diameter) of the magnetic field gradient concentrating particles is    about 300 μm.-   65. The method of any of paragraphs 57-64, wherein at least 50% area    or higher of said at least a portion of the magnetic capture surface    comprises the magnetic field gradient concentrating particles    distributed thereon.-   66. The method of any of paragraphs 57-65, wherein efficiency of    separating the magnetic particles from the fluid is increased by at    least about 50% (including, e.g., at least about 60%, at least about    70%, at least about 80%, at least about 90%) or more, as compared to    the efficiency in the absence of the magnetic field concentrating    particles.-   67. The method of any of paragraphs 57-66, wherein efficiency of    separating the magnetic particles from the fluid is increased by at    least about 1.1-fold (including, e.g., at least about 1.5-fold, at    least about 2-fold, at least about 3-fold, at least about 4-fold) or    more, as compared to the efficiency in the absence of the magnetic    field concentrating particles.-   68. The method of any of paragraphs 57-67, wherein a local magnetic    field gradient experienced by the magnetic particles is increased by    at least about 30% or higher (including, e.g., at least about 40%,    at least about 50%, at least about 60%, at least about 70%, at least    about 80%, at least about 90%, or higher), as compared to that    experienced by the magnetic particles in the absence of the magnetic    field concentrating particles.-   69. The method of any of paragraphs 57-68, wherein a local magnetic    field gradient experienced by the magnetic particles is increased by    at least about 1.1-fold or higher (including, e.g., at least about    1.5-fold, at least about 2-fold, at least about 3-fold, at least    about 4-fold, at least about 5-fold, at least about 10-fold, at    least about 50-fold, at least about 100-fold, at least about    150-fold or higher), as compared to that experienced by the magnetic    particles in the absence of the magnetic field concentrating    particles.-   70. The method of any of paragraphs 57-69, wherein the magnetic    capture surface forms at least part of a magnetic separation    chamber.-   71. The method of paragraph 70, wherein the magnetic separation    chamber comprises or is a channel, a microfluidic channel, a sample    well, a microtiter plate, a slide (e.g., a glass slide), a flask    (e.g., a tissue culture flask), a tube, a nanotube, a fiber, a    filter, a membrane, a scaffold, an extracorporeal device, a mixer, a    hollow fiber, or any combinations thereof.-   72. The method of any of paragraphs 57-67, wherein the fluid is    flowed through the magnetic separation chamber at a flow rate of    about 1 ml/hr to about 10 L/hr.-   73. The method of any of paragraphs 57-72, wherein the fluid is a    biological fluid obtained or derived from a subject, a fluid or    specimen obtained from an environmental source, a fluid from a cell    culture, a microbe colony, or any combinations thereof.-   74. The method of paragraph 73, wherein the fluid is a biological    fluid selected from blood, plasma, cord blood, serum, lactation    products, amniotic fluids, sputum, saliva, urine, semen,    cerebrospinal fluid, bronchial aspirate, bronchial lavage aspirate    fluid, perspiration, mucus, liquefied stool sample, synovial fluid,    peritoneal fluid, pleural fluid, pericardial fluid, lymphatic fluid,    tears, tracheal aspirate, a homogenate of a tissue specimen, or any    mixtures thereof.-   75. The method of paragraph 73, wherein the fluid is a fluid or    specimen obtained from an environmental source selected from a fluid    or specimen obtained or derived from food products, food produce,    poultry, meat, fish, beverages, dairy product, water (including    wastewater), ponds, rivers, reservoirs, swimming pools, soils, food    processing and/or packaging plants, agricultural places,    hydrocultures (including hydroponic food farms), pharmaceutical    manufacturing plants, animal colony facilities, beer brewing, or any    combinations thereof.-   76. The method of any of paragraphs 57-75, wherein the magnetic    particles are paramagnetic or superparamagnetic particles.-   77. The method of any of paragraphs 57-76, wherein the magnetic    particles are magnetic particles adapted or functionalized to bind a    target selected from the group consisting of cells, proteins,    nucleic acids, microbes, small molecules, chemicals, toxins, drugs,    and combinations thereof.-   78. The method of any of paragraphs 57-77, wherein the magnetic    particles are microbe-binding magnetic particles.-   79. The method of paragraph 78, wherein the microbe-binding magnetic    particles comprise on their surface microbe-binding molecules.-   80. The method of paragraph 79, wherein the microbe-binding molecule    is selected from the group consisting of opsonins, lectins,    antibodies and antigen binding fragments thereof, proteins,    peptides, peptidomimetics, carbohydrate-binding proteins, nucleic    acids, carbohydrates, lipids, steroids, hormones, lipid-binding    molecules, cofactors, nucleosides, nucleotides, nucleic acids,    peptidoglycan, lipopolysaccharide-binding proteins, small molecules,    and any combination thereof.-   81. The method of paragraph 79 or 80, wherein the microbe-binding    molecule comprises at least a microbial-binding portion of C-type    lectins, collectins, ficolins, receptor-based lectins, lectins from    the shrimp Marsupenaeus japonicas, non-C-type lectins,    lipopolysaccharide (LPS)-binding proteins, endotoxin-binding    proteins, peptidoglycan-binding proteins, or any combinations    thereof.-   82. The method of any of paragraphs 79-81, wherein the    microbe-binding molecule is selected from the group consisting of    mannose-binding lectin (MBL), surfactant protein A, surfactant    protein D, collectin 11, L-ficolin, ficolin A, DC-SIGN, DC-SIGNR,    SIGNR1, macrophage mannose receptor 1, dectin-1, dectin-2, lectin A,    lectin B, lectin C, wheat germ agglutinin, CD14, MD2,    lipopolysaccharide-binding protein (LBP), limulus anti-LPS factor    (LAL-F), mammalian peptidoglycan recognition protein-1 (PGRP-1),    PGRP-2, PGRP-3, PGRP-4, C-reactive protein (CRP), or any    combinations thereof.-   83. The method of any of paragraphs 78-82, wherein the    microbe-binding molecules are attached to the microbe-binding    magnetic particles via a linker.-   84. The method of paragraph 83, wherein the N-terminus of the linker    comprises an amino acid sequence of AKT (alanine, lysine,    threonine).-   85. The method of paragraph 83 or 84, wherein the linker is a    peptide linker.-   86. The method of any of paragraphs 83-85, wherein the linker    comprises an Fc portion of an immunoglobulin.-   87. The method of any of paragraphs 79-86, wherein the    microbe-binding molecule is selected from the group consisting of    MBL (mannose binding lectin), FcMBL (IgG Fc fused to mannose binding    lectin), AKT-FcMBL (IgG Fc-fused to mannose binding lectin with the    N-terminal amino acid tripeptide of sequence AKT (alanine, lysine,    threonine)), and any combination thereof.-   88. The method of any of paragraphs 79-87, wherein the    microbe-binding molecule comprises an amino acid sequence selected    from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID    NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, and any combination    thereof.-   89. The method of any of paragraphs 77-88, wherein the method    captures at least one target from the fluid.-   90. A method of capturing at least one target from a fluid    comprising:    -   introducing a fluid comprising target-binding magnetic particles        to a magnetic separation chamber in the presence of a magnetic        field gradient (a gradient of a magnetic field), wherein at        least a portion of a fluid-contact surface of the magnetic        separation chamber comprises magnetic field gradient        concentrating particles distributed thereon and aligned along        with magnetic flux lines of the magnetic field,    -   wherein the magnetic field gradient concentrating particles act        as local magnetic field gradient concentrators, thereby        attracting at least a portion of target-bound target-binding        magnetic particles to the magnetic field gradient concentrating        particles in the presence of the magnetic field gradient.-   91. The method of paragraph 90, wherein the magnetic field gradient    concentrating particles form magnetic micro- or nano-structures on    said at least a portion of the fluid-contact surface of the magnetic    separation chamber.-   92. The method of paragraph 1, 57, or 90, wherein the magnetic field    gradient concentrating particles are treated to reduce or inhibit    non-specific interaction between the magnetic field gradient    concentrating particles and said at least one target.-   93. The paragraph of paragraph 92, wherein the magnetic field    gradient concentrating particles are treated with a blocking agent.-   94. The paragraph of paragraph 93, wherein the blocking agent    comprises a lubricant (e.g., but not limited to silicone and/or    mold-release agent), a polymer (e.g., but not limited to    silicon-based polymer such as polydimethylsiloxane (PDMS)), milk    proteins, bovine serum albumin, blood serum, whole blood, or a    combination of two or more thereof.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. In one respect,the present invention relates to the herein described compositions,methods, and respective component(s) thereof, as essential to theinvention, yet open to the inclusion of unspecified elements, essentialor not (“comprising”). In some embodiments, other elements to beincluded in the description of the composition, method or respectivecomponent thereof are limited to those that do not materially affect thebasic and novel characteristic(s) of the invention (“consistingessentially of”). This applies equally to steps within a describedmethod as well as compositions and components therein. In otherembodiments, the inventions, compositions, methods, and respectivecomponents thereof, described herein are intended to be exclusive of anyelement not deemed an essential element to the component, composition ormethod (“consisting of”).

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used to described the present invention,in connection with percentages means±5%. When “0%” is used to describethe amount of a component, it is understood that this includessituations where only trace amounts of the component are present.

All patents, patent applications, and publications identified in thisdocument are expressly incorporated herein by reference for the purposeof describing and disclosing, for example, the methodologies describedin such publications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

EXAMPLES

The following examples are not intended to limit the scope of theinvention, but are rather intended to be exemplary of certainembodiments.

Example 1. Integration of Ferromagnetic Particles in a MicrofluidicDevice to Increase Throughput and/or Separation Efficiency of the Device

This Example describes a method to increase magnetic separationefficiency of an organ-on-chip device (e.g., a biospleen device asdescribed in the International Patent Application Nos. WO2012135834; WO2011091037; and WO 2007044642, the contents of each of which areincorporated herein by reference) using ferromagnetic particles trappedin the channels or coating fluid-contact surface of the channels. Theferromagnetic particles-integrated in the organ-on-chip devices (e.g., abiospleen device) can be used for high-throughput cleansing of anyfluid, e.g., blood, other biological fluids, water, food or any otherliquid that can flow.

The method involves ferromagnetic beads distributed or magneticallytrapped on fluid-contact surfaces of an organ-on-chip device (e.g.,either in the saline channel and blood channel of the biospleen device).To do this, for example, the ferromagnetic particles (˜10 mg to 500 mg)added to 10˜50 mL of either buffer or ethanol were introduced using aperistaltic pump into the channel(s) of the organ-on-chip device (e.g.,a biospleen device) where the magnet(s) were placed outside in proximityto the channel(s) of the organ-on-chip device to trap the ferromagneticparticles, e.g., onto the surface of the channel(s) while buffer orethanol containing the ferromagnetic particles was continuously flowingthrough the channel(s) of the device. In some embodiments where abiospleen device (e.g., described in WO2012135834), the magnet(s) wereplaced on the top (e.g., the saline channel) or at the bottom (e.g., theblood channel) of the device to trap the ferromagnetic particles ontothe surface of the channel.

Once the ferromagnetic particles were trapped inside the channel of thedevice, the magnet(s) were manipulated to distribute those ferromagneticparticles uniformly over the fluid-contact surface area of the channel.Once the ferromagnetic particles were dispersed, the magnets (e.g.,permanent magnets) were located either on one or both sides of thedevice. For example, magnet(s) were placed on the top and/or at thebottom of the biospleen device.

When more than one magnets are placed facing each other, the magnetsattract and pull each other. The ferromagnetic particles trapped insidethe channels become magnetized (saturated with the magnetic flux) andaligned along the magnetic flex lines diverging from the magnets on oneside (e.g., on the bottom) and converging toward the magnets on theopposing side (e.g., on the top). Because the ferromagnetic particlesare aligned vertically along the magnetic flux lines, they do not clogthe channels if, for example, less than <900 mg of ferromagneticparticles are loaded in a biospleen device.

Accordingly, different amount of the ferromagnetic particles (10 mg to500 mg) were distributed or magnetically trapped on surface(s) of anorgan-on-chip device (e.g., a biospleen device). The magnetic isolationefficiency was measured, for example, using microbes (e.g., S. aureus)captured by small microbe-binding magnetic particles (e.g., 50 nm FcMBLmagnetic beads) at a flow rate of 2 L/h to correlate the amount of theferromagnetic particles with the isolation efficiency. The ferromagneticparticles and the FcMBL magnetic beads were collected from the deviceafter removing magnets from the devices.

In some embodiments, different amount of the ferromagnetic particles(300 mg to 900 mg) were loaded in the biospleen device and the magneticisolation efficiency of S. aureus bound with 128 nm FcMBL magnetic beadsflowing at 2 L/h was measured. FIG. 2 shows that the isolationefficiency increased and plateaued at about 500 mg of the ferromagneticparticles. FIG. 3 shows that the ferromagnetic particles integrated inthe biospleen device enabled separation of pathogens bound to 50 nmbeads with over 99% efficiency at a flow rate of 1 L/h, which representsmore than a five-fold increase in isolation efficiency compared to thebiospleen device without the ferromagnetic particles.

The magnetic beads that captured target molecules (e.g., pathogens) andwere trapped by or attracted to the ferromagnetic particles can bereleased from the ferromagnetic particles, for example, by pipetting ordemagnetizing the ferromagnetic particles, e.g., with a demagnetizer.Therefore, the target molecules that bound to the magnetic beads can besubjected to subsequent analysis, such as ELISA or PCA, withoutinterference from the ferromagnetic particles.

Example 2. Integration of Ferromagnetic Particles in a MicrofluidicDevice for Depletion of Microbes (e.g., Pathogens) from Cord Blood

One of the challenges in cryopreservation of cord blood is that 5-7% ofcord blood samples are contaminated by pathogens (mostly E. coli), whichpotentially cause adverse effects on stem cells preserved in cord blood.Thus, treating cord blood to remove pathogenic contaminants is abeneficial step prior to a cryopreservation process.

As a proof of concept, 3-6 cfu of S. aureus and E. coli (Bioball®) werespiked into 10 mL of cord blood. The pathogen-containing cord blood wasmixed with FcMBL magnetic beads and then introduced into a ferromagneticparticle-integrated biospleen device (as described in Example 1) for 5hours at a flow rate of 20 mL/h. The method to prime the biospleendevice with ferromagnetic particles is the same as described inExample 1. Samples (250 uL) were taken at 0, 2, 4, and 5 hour-timepoints and then inoculated into blood culture vials, followed by 5-dayculture at 37° C.

After 2-5 days of culture at 37° C., the blood culture vials wasexamined to determine if they turned turbid or remained clear. A turbidblood culture is indicative of at least one pathogen remained in thecord blood samples that has flown through the biospleen device. FIG. 4shows that the pathogens initially spiked into the cord blood sampleswere successfully removed by the method described above, as evidenced bya clear blood culture even when the sample was treated with theferromagnetic particles-integrated biospleen device for only 2 hours,whereas the control cord blood consistently contained pathogens over 5hours. The control cord blood refers to cord blood flowing through abiospleen device without target-binding magnetic particles.

Example 3. Enhanced Magnetic Depletion Efficiency of Clinical IsolatePathogens in Eppendorf Tubes, Combining 50 nm Microbe-Binding MagneticBeads and Ferromagnetic Beads

One of the issues to address is to expand a range of microbes (e.g.,pathogens) that bind FcMBL magnetic beads so that they can be detectedeither by ELISA or microfluidic devices. Smaller magnetic beadsgenerally provide a higher binding efficiency. However, smaller beadsgenerate weak magnetic forces; thus, the conventional methods are notcapable of efficiently separate small magnetic beads from a fluidsample. The methods described herein provide enhanced magnetic forcegradient for separating magnetic particles, including smaller magneticparticles that cannot be efficiently separated by the conventionalmagnetic separation methods, from a fluid sample. Thus, smaller magneticparticles can be used to bind target molecules with the methodsdescribed herein.

In this Example, the RS218 clinical isolate of E. coli was incubatedwith 50 nm FcMBL magnetic beads for 20 min in TBST Ca⁺⁺ 5 mM, and thenferromagnetic particles (10-500 mg) were added into the solution,followed by magnetic depletion using magnet(s).

FIG. 5 shows that the magnetic depletion assay without addition offerromagnetic particles yielded about 30% magnetic isolation efficiencyeven in TBST Ca′. However, with the addition of ferromagnetic particlesinto the solution, >99% magnetic depletion efficiency was achieved withRS218 captured by 50 nm FcMBL magnetic beads.

Example 4. Enhanced Magnetic Depletion Efficiency of Clinical IsolatePathogens in 96-Well Plates Combining 50 nm Microbe-Binding MagneticBeads and Ferromagnetic Beads

The multi-well plate platform provides a powerful technology thatenables high throughput analysis of samples employing robotics andautomation. While the conventional 96 well plate-based ELISA platformusing target-binding magnetic beads (Kingfisher™ or BioTek Inc.)provides capability to detect multiple target molecules simultaneously,but the platform is not able to achieve high binding efficiency ofsmaller target-binding magnetic beads (e.g. 50 nm) due to their inherentweak magnetic force.

This Example shows distributing or dispersing ferromagnetic particles ona fluid-contact surface of a magnetic separation chamber significantlyincrease the capture efficiency of pathogens that have not been depletedby the conventional 96-well plate-based methods using the smallermicrobe-binding magnetic particles alone (without ferromagneticparticles).

The RS218 clinical isolate of E. coli was incubated with 50 nm FcMBLmagnetic beads for a period of time in TBST Ca⁺⁺ 5 mM. The ferromagneticparticles (e.g., about 10 mg to 500 mg by weight in total and theparticles were approximately 300 μm in diameter) were first added toeach well to form a ferromagnetic structure or layer, followed by theaddition of the fluid sample comprising the RS128 and FcMBL magneticbeads. The mixture was then followed by magnetic depletion in different96-well plate-based platform formats using magnet(s). FIG. 6A shows thatthe method described herein can be adapted for use in a 96-well deepwell plate (e.g., from KingFisher®). The movable plate head has a 96-tipcomb, where each tip of the comb holds and protects a magnet during themagnetic separation process. The ferromagnetic particles added in deepwells formed 2D or 3D nano- or micro-structure on the fluid-contactsurface of the tip when the movable plate head (with the magnetic tipcomb) was brought close to the deep wells. Incubating the RS218 clinicalisolate sample comprising the FcMBL magnetic beads in the presence ofthe formed ferromagnetic structures yielded over 99% capture efficiencyof RS218 E. coli bound with 50 nm FcMBL magnetic beads whereas nomicrobe depletion was observed when the plate was used without theferromagnetic particles.

FIG. 6B shows that the method described herein can also be adapted foruse in a 96-well conventional plate placed on a magnetic plate holderand a shaker. The ferromagnetic particles added in the wells formed 2Dor 3D nano- or micro-structure at least at the bottom surface of thewells where the magnets are located. Shaking the RS218 clinical isolatesample comprising the FcMBL magnetic beads for about 20 min at 700 rpmin the presence of the formed ferromagnetic structures yielded over 99%capture efficiency of RS218 E. coli bound with 50 nm FcMBL magneticbeads whereas no microbe depletion was observed when the plate was usedwithout the ferromagnetic particles.

FIG. 6C shows that the method described herein can also be adapted foruse in a 96-well conventional plate placed on a magnetic plate holderand a rotating mixer. The ferromagnetic particles added in the wellsformed 2D or 3D nano- or micro-structure at least at the bottom surfaceof the wells where the magnets are located. Rotating the RS218 clinicalisolate sample comprising the FcMBL magnetic beads at 10 rpm in thepresence of the formed ferromagnetic structures yielded over 99% captureefficiency of RS218 E. coli bound with 50 nm FcMBL magnetic beadswhereas no microbe depletion was observed when the plate was usedwithout the ferromagnetic particles.

FIG. 6D shows the depletion efficiency of RS218 E. coli bound on 50 nmFcMBL magnetic beads using different 96-well plate platforms as shown inFIGS. 6A-6C with and without the ferromagnetic particles. All threedifferent capture conditions yielded over 90% depletion efficiency of 50nm bead bound RS218 E. coli when the ferromagnetic particles arepresent.

Example 5. Improving Specificity of Magnetic Separation by BlockingMagnetic Field Gradient Concentrating Particles to Reduce Non-SpecificInteraction

In some embodiments, the magnetic field gradient concentrating particlescan have non-specific interaction with target molecule(s) to becaptured, removed, or separated from a fluid. To reduce or inhibit suchnon-specific interaction, the magnetic field gradient concentratingparticles can be pre-treated. By way of example only, ferromagnetic ironpowder (as an example of the magnetic field gradient concentratingparticles) has non-specific interaction with microbes or pathogens. Inthis Example, iron powder was treated with different blocking agents(e.g., but not limited to, mold-release agent such as silicone spray,PDMS, milk, and human whole blood) to evaluate effects of each blockingagent on pathogen depletion efficiency with or without target-bindingmagnetic particles (e.g., 50 nm FcMBL-coated magnetic particles). Insome embodiments, iron powder was treated by contacting with differentblocking agents for about 20 min (with mixing as desired). The ironpowder, upon contact with a blocking agent, was then washed with anappropriate buffer (e.g., at least twice or more). The iron powder(e.g., about 0.5 g) treated with different blocking reagents wasincubated with microbes of interest (e.g., S. aureus; 10³ CFU/mL) spikedin TBST buffer (without 50 nm FcMBL-coated magnetic particles), and themicrobial depletion efficiency was then measured. FIG. 7 (data withoutFcMBL-coated magnetic particles) shows that the tested blocking reagentsefficiently inhibited or reduced non-specific interaction between ironpowder and microbes or pathogens.

The blocked iron powder was then used in combination with 50 nmFcMBL-coated magnetic particles to assess microbial depletionefficiency. For example, microbes or pathogens (e.g., S. aureus) inabout 1 mL of TBST were first contacted with about 50 μL of 50 nmFcMBL-coated magnetic particles to allow the microbes to bind to theFcMBL-coated magnetic particles. The microbe-bound FcMBL-coated magneticparticles (e.g., S. aureus-bound FcMBL-coated magnetic particles) werethen depleted or removed from the fluid or sample by magnetic fieldgradients enhanced by the addition of the blocked iron powder. FIG. 7(data with FcMBL-coated magnetic particles) shows that most of the S.aureus-bound FcMBL-coated magnetic particles were depleted or removedfrom the fluid or sample by magnetic field gradients enhanced by theaddition of the blocked iron powder.

This Example shows that selectivity of capturing or separating targetmolecules or cells from a fluid can be significantly improved by (i)reducing non-specific interaction between magnetic field gradientconcentrating particles (e.g., iron powder) and target molecules to beremoved (e.g., microbes), and (ii) using a selective target-bindingmagnetic particles to capture the target molecules. In some embodiments,the selective target-binding magnetic particles can be so small in sizethat they are not efficiently separated by a conventional magneticseparator, e.g., due to a too weak magnetic moment, because the methods,devices, kits, and/or solid substrates described herein provide highefficiency of magnetic separation, even using small target-bindingmagnetic particles.

An exemplary approach of reducing non-specific interaction betweenmagnetic field gradient concentrating particles and target molecules tobe removed can comprise treating the magnetic field gradientconcentrating particles with a blocking agent. Examples of the blockingagent can include, but are not limited to, a lubricant (e.g., but notlimited to silicone and/or mold-release agent), a polymer (e.g., but notlimited to silicon-based polymer such as polydimethylsiloxane (PDMS)),milk proteins, bovine serum albumin, blood serum, whole blood, and acombination of two or more thereof.

What is claimed is:
 1. A method of capturing at least one target from afluid comprising: introducing a fluid and target-binding magneticparticles to a magnetic separation chamber of a device in the presenceof a magnetic field gradient (a gradient of a magnetic field), whereinthe target-binding magnetic particles are magnetic particles adapted tobind a target, wherein the device comprises a magnetic separationchamber, wherein at least a portion of a fluid-contact surface of themagnetic separation chamber comprises magnetic field gradientconcentrating particles distributed thereon, wherein magnetic fieldgradient concentrating particles are substantially aligned alongmagnetic flux lines of the magnetic field in presence of a magneticfield gradient, and wherein the magnetic field gradient concentratingparticles act as local magnetic field gradient concentrators in presenceof the magnetic field gradient.
 2. The method of claim 1, wherein thetarget is selected from the group consisting of cells, proteins, nucleicacids, microbes, small molecules, chemicals, toxins, drugs, andcombinations thereof.
 3. The method of claim 1, wherein thetarget-binding magnetic particles are microbe-binding magnetic particlesand comprise on their surface microbe-binding molecules.
 4. The methodof claim 3, wherein the microbe-binding molecule is selected from thegroup consisting of proteins, peptides, peptidomimetics, nucleic acids,carbohydrates, lipids, steroids, hormones, lipid-binding molecules,cofactors, nucleosides, nucleotides, nucleic acids, peptidoglycan, smallmolecules, and any combination thereof.
 5. The method of claim 3,wherein the microbe-binding molecule is selected from the groupconsisting of carbohydrate-binding proteins, lipopolysaccharide-bindingproteins, and any combination thereof.
 6. The method of claim 3, whereinthe microbe-binding molecule is selected from the group consisting ofopsonins, lectins, and any combination thereof.
 7. The method of claim3, wherein the microbe-binding molecule comprises at least amicrobial-binding portion of C-type lectins, collectins, ficolins,receptor-based lectins, lectins from the shrimp Marsupenaeus japonicas,non-C-type lectins, lipopolysaccharide (LPS)-binding proteins,endotoxin-binding proteins, peptidoglycan-binding proteins, or anycombinations thereof.
 8. The method of claim 3, wherein themicrobe-binding molecule is selected from the group consisting ofmannose-binding lectin (MBL), surfactant protein A, surfactant proteinD, collectin 11, L-ficolin, ficolin A, DC-SIGN, DC-SIGNR, SIGNR1,macrophage mannose receptor 1, dectin-1, dectin-2, lectin A, lectin B,lectin C, wheat germ agglutinin, CD14, MD2, lipopolysaccharide-bindingprotein (LBP), limulus anti-LPS factor (LAL-F), mammalian peptidoglycanrecognition protein-1 (PGRP-1), PGRP-2, PGRP-3, PGRP-4, C-reactiveprotein (CRP), or any combinations thereof.
 9. The method of claim 3,wherein the microbe-binding molecule is selected from the groupconsisting of MBL (mannose binding lectin), FcMBL (IgG Fc fused tomannose binding lectin), AKT-FcMBL (IgG Fc-fused to mannose bindinglectin with the N-terminal amino acid tripeptide of sequence AKT(alanine, lysine, threonine)), and any combination thereof.
 10. Themethod of claim 3, wherein the microbe-binding molecule comprises anamino acid sequence selected from SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8,and any combination thereof.
 11. The method of claim 1, wherein thefluid is a biological fluid obtained or derived from a subject, a fluidor specimen obtained from an environmental source, a fluid from a cellculture, a microbe colony, or any combinations thereof.
 12. The methodof claim 1, wherein the fluid comprises the target-binding magneticparticles.
 13. The method of claim 1, wherein the magnetic fieldgradient concentrating particles form magnetic micro- or nano-structureson said at least a portion of the fluid-contact surface of the magneticseparation chamber.
 14. The method of claim 1, wherein the magneticseparation chamber comprises a channel, a microfluidic channel, a samplewell, a microtiter plate, a slide, a flask, a tube, a nanotube, a fiber,a filter, a membrane, a scaffold, an extracorporeal device, a mixer, ahollow fiber, or any combinations thereof.
 15. The method of claim 1,wherein the magnetic field gradient concentrating particles are amixture of different sized magnetic field gradient concentratingparticles.
 16. The method of claim 1, wherein the target-bindingmagnetic particles are paramagnetic particles.
 17. The method of claim1, wherein the magnetic separation chamber comprises a source fluidchannel and a collection fluid channel connected to each other by aplurality of transfer channels.
 18. The method of claim 17, wherein theplurality of transfer channels is oriented substantially perpendicularto the source channel and the collection channel.
 19. The method ofclaim 17, wherein the device further comprises a first magnetic sourcepositioned in close proximity to the collection channel.
 20. The methodof claim 18, wherein the device further comprises a second magneticsource positioned in close proximity to the source channel.